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Nicole Appel

Publications and source records attributed to Nicole Appel.

5 recordsLinked to original sources
PubMed2005

Efficient rescue of hepatitis C virus RNA replication by trans-complementation with nonstructural protein 5A.

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.

Cell Line, Tumor↗
PubMed2005

Mutational analysis of hepatitis C virus nonstructural protein 5A: potential role of differential phosphorylation in RNA replication and identification of a genetically flexible domain.

Nonstructural protein 5A of the hepatitis C virus (HCV) is a highly phosphorylated molecule implicated in multiple interactions with the host cell and most likely involved in RNA replication. Two phosphorylated variants of NS5A have been described, designated according to their apparent molecular masses (in kilodaltons) as p56 and p58, which correspond to the basal and hyperphosphorylated forms, respectively. With the aim of identifying a possible role of NS5A phosphorylation for RNA replication, we performed an extensive mutation analysis of three serine clusters that are involved in phosphorylation and hyperphosphorylation of NS5A. In most cases, alanine substitutions for serine residues in the central cluster 1 that enhanced RNA replication to the highest levels led to a reduction of NS5A hyperphosphorylation. Likewise, several highly adaptive mutations in NS4B, which is also part of the replication complex, resulted in a reduction of NS5A hyperphosphorylation too, arguing that alterations of the NS5A phosphorylation pattern play an important role for RNA replication. On the other hand, a deletion encompassing all highly conserved serine residues in the C-terminal region of NS5A that are involved in basal phosphorylation did not significantly affect RNA replication but reduced formation of p56. This region was found to tolerate even large insertions with only a moderate effect on replication. Based on these results, we propose a model of the role of NS5A phosphorylation in the viral life cycle.

Amino Acid Substitution↗
PubMed2004

Structure and function of the membrane anchor domain of hepatitis C virus nonstructural protein 5A.

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a membrane-associated, essential component of the viral replication complex. Here, we report the three-dimensional structure of the membrane anchor domain of NS5A as determined by NMR spectroscopy. An alpha-helix extending from amino acid residue 5 to 25 was observed in the presence of different membrane mimetic media. This helix exhibited a hydrophobic, Trprich side embedded in detergent micelles, while the polar, charged side was exposed to the solvent. Thus, the NS5A membrane anchor domain forms an in-plane amphipathic alpha-helix embedded in the cytosolic leaflet of the membrane bilayer. Interestingly, mutations affecting the positioning of fully conserved residues located at the cytosolic surface of the helix impaired HCV RNA replication without interfering with the membrane association of NS5A. In conclusion, the NS5A membrane anchor domain constitutes a unique platform that is likely involved in specific interactions essential for the assembly of the HCV replication complex and that may represent a novel target for antiviral intervention.

Amino Acid Sequence↗