Conserved Determinants for Membrane Association of Nonstructural Protein 5A fromHepatitis C Virus and Related Viruses

Abstract

Nonstructural protein 5A (NS5A) is a membrane-associated essential component of the hepatitis C virus (HCV) replication complex. An N-terminal amphipathic alpha helix mediates in-plane membrane association of HCV NS5A and at the same time is likely involved in specific protein-protein interactions required for the assembly of a functional replication complex. The aim of this study was to identify the determinants for membrane association of NS5A from the related GB viruses and pestiviruses. Although primary amino acid sequences differed considerably, putative membrane anchor domains with amphipathic features were predicted in the N-terminal domains of NS5A proteins from these viruses. Confocal laser scanning microscopy, as well as membrane flotation analyses, demonstrated that NS5As from GB virus B (GBV-B), GBV-C, and bovine viral diarrhea virus, the prototype pestivirus, display membrane association characteristics very similar to those of HCV NS5A. The N-terminal 27 to 33 amino acid residues of these NS5A proteins were sufficient for membrane association. Circular dichroism analyses confirmed the capacity of these segments to fold into alpha helices upon association with lipid-like molecules. Despite structural conservation, only very limited exchanges with sequences from related viruses were tolerated in the context of functional HCV RNA replication, suggesting virus-specific interactions of these segments. In conclusion, membrane association of NS5A by an N-terminal amphipathic alpha helix is a feature shared by HCV and related members of the family Flaviviridae. This observation points to conserved roles of the N-terminal amphipathic alpha helices of NS5A in replication complex formation.The family Flaviviridae comprises the genera Flavirus, Hepacivirus, and Pestivirus, as well as the as-yet-unassigned GB virus A (GBV-A), GBV-B, and GBV-C. These enveloped positivestrand RNA viruses express their structural and nonstructural proteins via translation of a single long open reading frame (30). Hepatitis C virus (HCV) is the sole member of the genus Hepacivirus and a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma, affecting an estimated 3% of the world's population (44). GB viruses and pestiviruses are more closely related to HCV than the classical flaviviruses. The availability of efficient cell culture systems and animal models makes these related viruses attractive in vitro and in vivo models for HCV. GBV-B, the closest relative of HCV, was recovered from a tamarin inoculated with blood from a surgeon suffering from acute hepatitis (16, 54) and causes acute and chronic hepatitis in New World monkeys, which are believed to represent the natural hosts (10). Subsequent attempts to isolate GBV-B sequences from humans have failed. By contrast, GBV-C persistently infects humans. However, the pathogenic relevance of GBV-C, if any, remains unclear. It does not cause hepatitis but has been associated with prolonged survival in human immunodeficiency virus-infected individuals (62-64). ...

Correlation between NS5A Dimerization and Hepatitis C Virus Replication

“…It has also been shown to interact with a large number of host proteins including CypA (cyclophilin A) (15)(16)(17)(18)(19)(20)(21)(22). NS5A is peripherally anchored to membranes by an N-terminal amphipathic helix (23)(24)(25)(26)(27). It is organized into three distinct domains, separated by two repetitive low complexity sequence stretches (28).…”

A comparative cell biological analysis reveals only limited functional homology between the NS5A proteins of hepatitis C virus and GB virus B

“…Both HCV and GBV-B NS5A possess an N-terminal amphipathic helix that mediates membrane association (Brass et al, 2007), and conserved cysteines that co-ordinate a zinc ion (Tellinghuisen et al, 2004). In the case of HCV both these elements are essential for HCV RNA replication; thus it is likely that, unlike the situation regarding perturbation of host-cell signalling, the RNA replication functions of the two NS5A proteins are conserved.…”

“…Both HCV and GBV-B NS5A proteins contain an N-terminal amphipathic helix reported to mediate membrane association (Brass et al, 2002(Brass et al, , 2007. To investigate if GBV-B NS5A exhibited the same subcellular distribution as HCV NS5A, cells were co-transfected with pSG5 vectors expressing the FLAG-tagged GBV-B NS5A and HCV NS5A and analysed by immunofluorescence and confocal microscopy.…”

“…A C-terminal tag was chosen so as not to interfere with the function of the N-terminal amphipathic helix present in GBV-B NS5A (Brass et al, 2007). Expression of the FLAG-tagged GBV-B NS5A was verified by Western blot analysis (Fig.…”

Interacción entre el virus de la inmunodeficiencia humana y el virus GB tipo-C durante el estado de co-infección

“…NS5A es reconocida como una proteína con capacidad para interactuar con múltiples blancos celulares. Teniendo en cuenta los estudios realizados para el VHC, NS5A se localiza en retículo endoplásmico gracias a que en su extremo amino-terminal se encuentra una hélice anfipática de interacción a membrana, siendo necesaria para la replicación viral 47,48 . Se han caracterizado tres dominios principales; el dominio I localizado en el extremo amino-terminal que presenta motivos de zinc para unión a ácidos nucleicos, mientras los dominios II y III contienen los sitios de hiperfosforilación y fosforilación basal, respectivamente 49 .…”