Hepatitis C virus nonstructural protein 4B: A journey into unexplored territory

Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland.
Reviews in Medical Virology (Impact Factor: 5.57). 03/2010; 20(2):117-29. DOI: 10.1002/rmv.640
Source: PubMed


Hepatitis C virus (HCV) is a positive-strand RNA virus that replicates its genome in a membrane-associated replication complex. Nonstructural protein 4B (NS4B) induces the specific membrane alteration, designated as membranous web (MW), that harbours this complex. HCV NS4B is an integral membrane protein predicted to comprise four transmembrane segments in its central part. The N-terminal part comprises two amphipathic alpha-helices of which the second has the potential to traverse the membrane bilayer, likely upon oligomerisation. The C-terminal part comprises a predicted highly conserved alpha-helix, a membrane-associated amphipathic alpha-helix and two reported palmitoylation sites. NS4B interacts with other viral nonstructural proteins and has been reported to bind viral RNA. In addition, it was found to harbour an NTPase activity. Finally, NS4B has recently been found to have a role in viral assembly. Much work needs to be done with respect to further dissecting these multiple functions as well as providing a refined membrane topology and complete structure of NS4B. Progress in this direction should yield important insights into the functional architecture of the HCV replication complex and may reveal new opportunities for antiviral intervention against a leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide.

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Available from: Francois Penin, Aug 25, 2014
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    • "A key viral protein involved in membrane remodelling events is NS4B (Egger et al., 2002). While the full structure of NS4B has yet to be solved, the protein is believed to possess two aminoterminal amphipathic helices, four central trans-membrane domains and two a-helices in the carboxy-terminal domain (reviewed by Gouttenoire et al., 2010a). NS4B self-interacts via multiple determinants, which is required for its function in vRF biogenesis (Gouttenoire et al., 2010b; Paul et al., 2011). "
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    ABSTRACT: Hepatitis C virus (HCV) represents a significant global health burden. Viral replication is thought to occur in close association with remodelled host cell membranes with non-structural protein 4B (NS4B) being a key player in this process. NS4B is a poorly characterized integral membrane protein, which has been reported to be palmitoylated at its carboxy-terminal end. In order to extend this observation and to establish a functional role for NS4B palmitolylation, we sought to determine the status of this post-translational modification when the protein was expressed as part of a functional viral replicase. We performed direct metabolic labelling and polyethylene glycol-maleimide palmitoylation reporter assays on NS4B expressed in cells containing subgenomic replicons and infectious viral RNA. In a vaccinia virus-based expression system NS4B palmitoylation was detected in a genotype-dependent manner. However, in spite of the high sensitivity of the methods used, no NS4B palmitoylation was found in physiologically more relevant systems. Thus, NS4B palmitoylation is most likely dispensable for HCV RNA replication.
    Full-text · Article · Mar 2015 · Journal of General Virology
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    • "This multi-functionality mostly stems from their ability to form diverse but higher ordered dimeric, heteromeric or oligomeric structures. There are many examples of such structures among the viral proteins that have been reported in the literature, including human immunodeficiency virus 1 (HIV-1) proteins (Rev, Vpr, Vif and Vpu) (Bernacchi et al., 2011; Bourbigot et al., 2005; Daugherty et al., 2010a, 2010b; Lu et al., 2010), Hepatitis C virus nonstructural protein 4B (Gouttenoire et al., 2010), Ebola virus VP40 (Hoenen et al., 2010), and large T-antigen (LT-Ag) of polyomaviruses (Cuesta et al., 2010; Foster and Simmons, 2010). Recently, agnoprotein of polyomaviruses was also shown to operate in this fashion. "
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    ABSTRACT: Agnoprotein is one of the key regulatory proteins of polyomaviruses, including JCV, BKV and SV40 and is required for a productive viral life cycle. We have recently reported that agnoprotein forms stable dimer/oligomers mediated by a predicted amphipathic α-helix, spanning amino acids (aa), 17 to 42. Deletion of the α-helix renders a replication incompetent virus. Here, we have further characterized this region by a systematic deletion and substitution mutagenesis and demonstrated that a Leu/Ile/Phe-rich domain, (spanning aa 28-39) within α-helix is indispensable for agnoprotein structure and function. Deletion of aa 30-37 severely affects the dimer/oligomer formation and stable expression of the protein. Mutagenesis data also indicate that the residues, 34-36, may be involved in regulation of the splicing events of JCV transcripts. Collectively, these data suggest that the Leu/Ile/Phe-rich domain plays critical roles in agnoprotein function and thus represents a potential target for developing novel therapeutics against JCV infections.
    Full-text · Article · Jun 2013 · Virology
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    • "This result supports the role of W38 and F41 in membrane association. Nonstructural viral proteins with hydrophobic sequences may transverse the membrane similar to polytopic integral membrane proteins: nonstructural proteins NS2 and NS4B of Hepatitis C virus (HCV) are integral polytopic membrane proteins (Gouttenoire et al., 2010; Yamaga and Ou, 2002). It is also known that viral nonstructural proteins, such as BMV 1a and semliki forest virus nsP1, interact with membranes monotopically as a peripheral membrane protein via an amphipathic a-helical peptide that enables their membrane integration or interaction (Liu et al., 2009; Salonen et al., 2005; Schwartz et al., 2004; Spuul et al., 2007). "
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    ABSTRACT: Positive-strand RNA viruses require host intracellular membranes for replicating their genomic RNAs. In this study, we determined the domains and critical amino acids in p27 of Red clover necrotic mosaic virus (RCNMV) required for its association with and targeting of ER membranes in Nicotiana benthamiana plants using a C-terminally GFP-fused and biologically functional p27. Confocal microscopy and membrane-flotation assays using an Agrobacterium-mediated expression system showed that a stretch of 20 amino acids in the N-terminal region of p27 is essential for the association of p27 with membranes. We identified the amino acids in this domain required for the association of p27 with membranes using alanine-scanning mutagenesis. We also found that this domain contains amino acids not critical for the membrane association but required for the formation of viral RNA replication complexes and negative-strand RNA synthesis. Our results extend our understanding of the multifunctional role of p27 in RCNMV replication.
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