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

2006 ◽  
Vol 81 (6) ◽  
pp. 2745-2757 ◽  
Author(s):  
Volker Brass ◽  
Zsuzsanna Pal ◽  
Nicolas Sapay ◽  
Gilbert Deléage ◽  
Hubert E. Blum ◽  
...  
Keyword(s):  
Amino Acid ◽  
Nonstructural Protein ◽  
Alpha Helix ◽  
Laser Scanning Microscopy ◽  
Membrane Association ◽  
Alpha Helices ◽  
Replication Complex ◽  
Diarrhea Virus ◽  
Amphipathic Alpha Helix ◽  
Nonstructural Protein 5A

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.

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

2009 ◽  
Vol 83 (12) ◽  
pp. 6257-6268 ◽  
Author(s):  
Jérôme Gouttenoire ◽  
Valérie Castet ◽  
Roland Montserret ◽  
Naveen Arora ◽  
Vincent Raussens ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Nonstructural Protein ◽  
Attenuated Total Reflection ◽  
Membrane Extraction ◽  
Phospholipid Bilayer ◽  
Membrane Association ◽  
Acceptor Site ◽  
Replication Complex ◽  
Α Helix

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 α-helix extending from aa 42 to 66. Attenuated total reflection infrared spectroscopy and glycosylation acceptor site tagging revealed that this amphipathic α-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.

scholarly journals Efficient Rescue of Hepatitis C Virus RNA Replication by trans-Complementation with Nonstructural Protein 5A

2005 ◽  
Vol 79 (2) ◽  
pp. 896-909 ◽  
Author(s):  
Nicole Appel ◽  
Ulrike Herian ◽  
Ralf Bartenschlager
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Functional Organization ◽  
Nonstructural Protein ◽  
Rna Replication ◽  
Hcv Rna ◽  
Replication Complex ◽  
Diarrhea Virus ◽  
Amino Terminal ◽  
Essential Components

ABSTRACT Studies of Hepatitis C virus (HCV) RNA replication have become possible with the development of subgenomic replicons. This system allows the functional analysis of the essential components of the viral replication complex, which so far are poorly defined. In the present study we wanted to investigate whether lethal mutations in HCV nonstructural genes can be rescued by trans-complementation. Therefore, a series of replicon RNAs carrying mutations in NS3, NS4B, NS5A, and NS5B that abolish replication were transfected into Huh-7 hepatoma cells harboring autonomously replicating helper RNAs. Similar to data described for the Bovine viral diarrhea virus (C. W. Grassmann, O. Isken, N. Tautz, and S. E. Behrens, J. Virol. 75:7791-7802, 2001), we found that only NS5A mutants could be efficiently rescued. There was no evidence for RNA recombination between helper and mutant RNAs, and we did not observe reversions in the transfected mutants. Furthermore, we established a transient complementation assay based on the cotransfection of helper and mutant RNAs. Using this assay, we extended our results and demonstrated that (i) inactivating NS5A mutations affecting the amino-terminal amphipathic helix cannot be complemented in trans; (ii) replication of the helper RNA is not necessary to allow efficient trans-complementation; and (iii) the minimal sequence required for trans-complementation of lethal NS5A mutations is NS3 to -5A, whereas NS5A expressed alone does not restore RNA replication. In summary, our results provide the first insight into the functional organization of the HCV replication complex.

The amphipathic alpha helices of the toxoplasma protein GRA2 mediate post-secretory membrane association

1998 ◽  
Vol 111 (15) ◽  
pp. 2171-2180 ◽  
Author(s):  
C. Mercier ◽  
M.F. Cesbron-Delauw ◽  
L.D. Sibley
Keyword(s):  
Amino Acids ◽  
Toxoplasma Gondii ◽  
Molecular Basis ◽  
Alpha Helix ◽  
Vacuolar Membrane ◽  
Membrane Association ◽  
Deletion Mutants ◽  
Cell Fractionation ◽  
Alpha Helices ◽  
Dense Granules

The Toxoplasma gondii protein GRA2 is secreted into the parasite-containing vacuole where it is rapidly and specifically targeted to a network of membranous tubules that connect with the vacuolar membrane. To examine the molecular basis of this association, we expressed an HA9 epitope-tagged form of GRA2 by stable transformation of Toxoplasma. GRA2-HA9 was correctly packaged inside the dense granules, secreted into the PV and targeted to the network, as shown by immunoelectron microscopy, immunofluorescence and cell fractionation. Expression of deletion mutants of GRA2-HA9 lacking either of two amphipathic alpha helices resulted in the production and secretion of soluble proteins which were unable to stably associate with the network. A mutant in which the amino acids of the first alpha helix were rearranged to a non-amphipathic pattern localized correctly to the network but failed to remained stably associated with the membrane. Collectively, these results demonstrate that targeting and membrane association occur by separate mechanisms and that the combination of both alpha helices is essential for stable localization of GRA2 to the network.

scholarly journals Impact of a baseline polymorphism on the emergence of resistance to the hepatitis C virus nonstructural protein 5a replication complex inhibitor, BMS-790052

Hepatology ◽  
2012 ◽  
Vol 55 (6) ◽  
pp. 1692-1699 ◽  
Author(s):  
Jin-Hua Sun ◽  
Donald R. O'Boyle II ◽  
Yan Zhang ◽  
Chunfu Wang ◽  
Peter Nower ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Nonstructural Protein ◽  
Replication Complex ◽  
Emergence Of Resistance ◽  
Nonstructural Protein 5A

scholarly journals Fine dissection of a nine amino acid glycoprotein epitope, a major determinant recognized by lymphocytic choriomeningitis virus-specific class I-restricted H-2Db cytotoxic T lymphocytes.

1988 ◽  
Vol 168 (2) ◽  
pp. 559-570 ◽  
Author(s):  
M B Oldstone ◽  
J L Whitton ◽  
H Lewicki ◽  
A Tishon
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Aromatic Amino Acids ◽  
Alpha Helix ◽  
Lymphocytic Choriomeningitis ◽  
Lymphocytic Choriomeningitis Virus ◽  
Specific Class ◽  
Viral Glycoprotein ◽  
Viral Epitope ◽  
Amphipathic Alpha Helix

We define a nine-amino acid (aa) sequence of VAL-GLU-ASN-PRO-GLY-GLY-TYR-CYS-LEU as a major epitope for immunologic recognition of lymphocytic choriomeningitis virus (LCMV) by H-2b-restricted CTL. The epitope was characterized using molecular genetics to bracket broadly and chemistry to precisely identify aa residues 278-286 of the viral glycoprotein. The epitope's composition is characteristic of a reverse (beta turn) but not an amphipathic alpha helix. A series of peptides with a single aa substitution in position 278 of VAL with other nonpolar (hydrophobic) amino acids (LEU, ILE, ALA, or GLY) coat targets that are recognized and lysed by CTL clones recognizing this epitope. In contrast, substitution of VAL with either large aromatic amino acids (that add bulk: PHE, TYR) or polar side chains (SER, THR) segregates CTL clones normally recognizing aa 278-286 into two groups, one that remains lytic (permissive) despite these changes and another that fails to lyse, indicating CTL can discriminate at a single aa. A change in charge at this position (VAL----ASP or GLU), in general, reduces CTL lysis while a change of VAL to LYS or ASN has minimal affect for four of the five CTL clones analyzed. CTL reactivity with the viral epitope is restricted by the Db but not the Kb of the murine MHC haplotype. A 16-aa peptide of Db that spans alpha 1 residues 37-52 blocks CTL lysis, whereas the corresponding Kb peptide that differs from Db in a single aa in position 50 does not.

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