Phenotypic analysis of NS5A variant from liver transplant patient with increased cyclosporine susceptibility

University of Wisconsin-Madison, Madison, WI, United States.
Virology (Impact Factor: 3.32). 02/2013; 436(2). DOI: 10.1016/j.virol.2012.11.018
Source: PubMed


Hepatitis C virus (HCV) replication is limited by cyclophilin inhibitors but it remains unclear how viral genetic variations influence susceptibility to cyclosporine (cyclosporine A, CsA), a cyclophilin inhibitor. In this study HCV from liver transplant patients was sequenced before and after CsA exposure. Phenotypic analysis of NS5A sequence was performed by using HCV sub genomic replicon to determine CsA susceptibility. The data indicates an atypical proline at position 328 in NS5A causes increases CsA sensitivity both in the context of genotype 1a and 1b residues. Point mutants mimicking other naturally occurring residues at this position also increased (Ala) or decreased (Arg) replicon sensitivity to CsA relative to the typical threonine (genotype 1a) or serine (genotype 1b) at this position. This work has implications for treatment of HCV by cyclophilin inhibitors.

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Available from: Israrul Ansari, May 12, 2015
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    • "To aid in the analysis of this data deluge, it is imperative that standardized annotations describing the characteristics of the isolated strains (metadata) are recorded, reported and associated with the corresponding sequence record. For example, interrogations into the emergence and seasonality of new infectious disease outbreaks can be conducted only if metadata describing the time and place of isolation is available (Bloom- Feshbach et al., 2013; Patel et al., 2013); identification of sequence substitutions that correlate with host specificity requires pathogen host species information (Shi and Hu, 2008); and investigations of sequence biomarkers that contribute to pathogen virulence and drug resistance can only be performed if the host's health status and medical history are available (Ansari et al., 2013; Kaminski et al., 2013; Schvoerer et al., 2013). Access to additional metadata will lead to increased data exploration and hypothesis generation that can then be subjected to 'wet lab' experiments to validate the observations, biochemically identify the mechanism of actions and "
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    ABSTRACT: The Virus Pathogen Resource (ViPR; and Influenza Research Database (IRD; have developed a metadata-driven Comparative Analysis Tool for Sequences (meta-CATS), which performs statistical comparative analyses of nucleotide and amino acid sequence data to identify correlations between sequence variations and virus attributes (metadata). Meta-CATS guides users through: selecting a set of nucleotide or protein sequences; dividing them into multiple groups based on any associated metadata attribute (e.g. isolation location, host species); performing a statistical test at each aligned position; and identifying all residues that significantly differ between the groups. As proofs of concept, we have used meta-CATS to identify sequence biomarkers associated with dengue viruses isolated from different hemispheres, and to identify variations in the NS1 protein that are unique to each of the 4 dengue serotypes. Meta-CATS is made freely available to virology researchers to identify genotype-phenotype correlations for development of improved vaccines, diagnostics, and therapeutics.
    Full-text · Article · Dec 2013 · Virology
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    • "Whilst two related crystal structures have been determined with different dimeric interfaces for the N terminus of NS5A (Tellinghuisen et al., 2005; Love et al., 2009), the protein is also likely to have regions of disorder, and the overall tertiary and quaternary structure of NS5A in the cell is unknown. There has been a focus on NS5A for some time as a possible mediator of IFN resistance (Enomoto et al., 1996) as the target of host-cell cyclophilin inhibitors (Anasri & Striker, 2012; Ansari et al., 2013; Membreno et al., 2013) and more selective antivirals (Lok et al., 2012), 3These authors contributed equally to this publication. "
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    ABSTRACT: NS5A has emerged as an important pharmacologic target for Hepatitis C Virus (HCV). Little is known though about the conformation of NS5A intracellularly or how NS5A inhibitors achieve the picomolar (pM) inhibition of viral replication. Here we present two structurally related small molecules, one that potently inhibits HCV replication and selects for resistance in NS5A and the other that is inactive. Resistance to this antiviral is greater in genotype 1a replicons than genotype 1b and maps to domain 1 of NS5A. Using a novel cell based assay that measures the intracellular proximity of fluorescent tags covalently attached to NS5A we show only the active antiviral specifically disrupts the close proximity of inter- and intra-molecular positions of NS5A. The active antiviral compound 1 causes a repositioning of both the amino and carboxy termini of NS5A including a disruption of the close approximation of the amino termini of two different NS5A molecules in a multi-molecular complex. Inter-molecular interactions are not disrupted in the presence of the active compound as NS5A remains in a multimeric state although it at least partially loses ER localization. This data provides the first study of how antivirals that select resistance in domain 1 of NS5A alter the cellular conformation of NS5A. This class of antiviral disrupts the close proximity of the amino termini of domain 1 in a NS5A complex, but also alters the conformation of domain 3 as well and leads to large aggregates of NS5A. Current models predict that a multicomponent cocktail of antivirals are needed to treat HCV infection so a mechanistic understanding of what each component does to the viral machinery is important.
    Full-text · Article · Aug 2013 · Journal of General Virology
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    ABSTRACT: Cyclophilins are peptidyl-prolyl cis/trans isomerases important in the proper folding of certain proteins. Mounting evidence supports varied roles of cyclophilins, either positive or negative, in the life cycles of diverse viruses, but the nature and mechanisms of these roles are yet to be defined. The potential for cyclophilins to serve as a drug target for antiviral therapy is evidenced by the success of non-immunosuppressive cyclophilin inhibitors (CPIs), including Alisporivir, in clinical trials targeting hepatitis C virus infection. In addition, as cyclophilins are implicated in the predisposition to, or severity of, various diseases, the ability to specifically and effectively modulate their function will prove increasingly useful for disease intervention. In this review, we will summarize the evidence of cyclophilins as key mediators of viral infection and prospective drug targets.
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