Phenotypic Characterization of Resistant Val
            <sup>36</sup>
            Variants of Hepatitis C Virus NS3-4A Serine Protease

Zhou, Yi; Bartels, Doug J.; Hanzelka, Brian L.; Müh, Ute; Wei, Yang; Chu, Hui‐May; Tigges, Ann M.; Brennan, Debra; Rao, B. Govinda; Swenson, Lora; Kwong, Ann D.; Lin, Chao

doi:10.1128/aac.00863-07

Cited by 90 publications

(83 citation statements)

References 50 publications

(129 reference statements)

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“…Many structure-activity-relationship (SAR) studies have been performed to evaluate the effect of different functional moieties on protease inhibition at positions P4-P1′ (11)(12)(13)(14)(15)(16)(17). Crystal structures have been determined of the NS3/4A protease domain bound to a variety of inhibitors as well as of several drug resistant protease variants, such as R155K and V36M (18,19). These data elucidate the molecular interactions of NS3/4A with inhibitors and the effect of specific drug resistance mutations on binding.…”

mentioning

confidence: 99%

Drug resistance against HCV NS3/4A inhibitors is defined by the balance of substrate recognition versus inhibitor binding

Romano

Ali

Royer

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

182

245

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Hepatitis C virus infects an estimated 180 million people worldwide, prompting enormous efforts to develop inhibitors targeting the essential NS3/4A protease. Resistance against the most promising protease inhibitors, telaprevir, boceprevir, and ITMN-191, has emerged in clinical trials. In this study, crystal structures of the NS3/4A protease domain reveal that viral substrates bind to the protease active site in a conserved manner defining a consensus volume, or substrate envelope. Mutations that confer the most severe resistance in the clinic occur where the inhibitors protrude from the substrate envelope, as these changes selectively weaken inhibitor binding without compromising the binding of substrates. These findings suggest a general model for predicting the susceptibility of protease inhibitors to resistance: drugs designed to fit within the substrate envelope will be less susceptible to resistance, as mutations affecting inhibitor binding would simultaneously interfere with the recognition of viral substrates.drug design | hepatitis C | substrate envelope D rug resistance is a major obstacle in the treatment of quickly evolving diseases. Hepatitis C virus (HCV) is a genetically diverse Hepacivirus of the Flaviviridae family infecting an estimated 180 million people worldwide (1). The viral RNA genome is translated as a single polyprotein and subsequently processed by host-cell and viral proteases into structural (C, E1, E2, and p7) and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins (2). The viral RNA-dependent RNA polymerase, NS5B, is inherently inaccurate and misincorporation of bases accounts for a very high mutation rate (3). While some mutations are neutral, others will alter the viability of the virus and propagate with varying efficiencies in each patient. Thus HCV infected individuals will develop a heterogeneous population of virus variants known as quasispecies (4). As patients begin treatment, the selective pressures of antiviral drugs will favor drug resistant variants (5). Therefore, an inhibitor must not only recognize one protein variant, but an ensemble of related enzymes. A detailed understanding of the atomic mechanisms of resistance is essential to effectively combat drug resistance against HCV antivirals.The essential HCV NS3/4A protease is an attractive therapeutic target responsible for cleaving at least four sites along the viral polyprotein. These sites share little sequence homology except for an acid at position P6, Cys or Thr at P1, and Ser or Ala at P1′ (Table S1). The first cleavage event at the 3-4A junction occurs in cis as a unimolecular process, while the remaining substrates are processed bimolecularly in trans. The NS3/4A protease also cleaves the human cellular targets TRIF and MAVS, which confounds the innate immune response to viral infection (6-8).Early drug design efforts were hampered by the relatively shallow, featureless architecture of the protease active site. The eventual observation of N-terminal product inhibition served as a stepping stone f...

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mentioning

confidence: 99%

Drug resistance against HCV NS3/4A inhibitors is defined by the balance of substrate recognition versus inhibitor binding

Romano

Ali

Royer

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

182

245

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“…As observed in other studies, acquiring resistance to an antiviral agent lowered strain fitness vis-à-vis the uninhibited wild-type levels (6,16,25). The acquisition of compensatory mutations occasionally restores replication capacity (7), and coselection for dependence on the antiviral chemical compounds has been documented (1,19).…”

Section: Discussionmentioning

confidence: 61%

“…To the best of our knowledge, this is the first report of a virus evolving a mechanism to productively utilize an antiviral agent to stimulate its fitness above the uninhibited wild-type level. The results of this study may be predictive of the types of resistant phenotypes that could be selected by antiviral agents that specifically target capsid assembly.While viruses often acquire resistance to antiviral agents, resistance mutants generally exhibit lower fitness than the wildtype strain in the absence of the inhibitor (6,16,17,25) and can develop a dependency on the antiviral agent (1, 19). However, the molecular mechanism of dependency rarely, if ever, involves the productive use of the antiviral agent to elevate fitness above the uninhibited wild-type level.…”

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confidence: 99%

“…While viruses often acquire resistance to antiviral agents, resistance mutants generally exhibit lower fitness than the wildtype strain in the absence of the inhibitor (6,16,17,25) and can develop a dependency on the antiviral agent (1, 19). However, the molecular mechanism of dependency rarely, if ever, involves the productive use of the antiviral agent to elevate fitness above the uninhibited wild-type level.…”

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Viral Adaptation to an Antiviral Protein Enhances the Fitness Level to Above That of the Uninhibited Wild Type

Cherwa

Sanchez-Soria

Wichman

et al. 2009

J Virol

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Viruses often evolve resistance to antiviral agents. While resistant strains are able to replicate in the presence of the agent, they generally exhibit lower fitness than the wild-type strain in the absence of the inhibitor. In some cases, resistant strains become dependent on the antiviral agent. However, the agent rarely, if ever, elevates dependent strain fitness above the uninhibited wild-type level. This would require an adaptive mechanism to convert the antiviral agent into a beneficial growth factor. Using an inhibitory scaffolding protein that specifically blocks X174 capsid assembly, we demonstrate that such mechanisms are possible. To obtain the quintuple-mutant resistant strain, the wild-type virus was propagated for approximately 150 viral life cycles in the presence of increasing concentrations of the inhibitory protein. The expression of the inhibitory protein elevated the strain's fitness significantly above the uninhibited wild-type level. Thus, selecting for resistance coselected for dependency, which was characterized and found to operate on the level of capsid nucleation. To the best of our knowledge, this is the first report of a virus evolving a mechanism to productively utilize an antiviral agent to stimulate its fitness above the uninhibited wild-type level. The results of this study may be predictive of the types of resistant phenotypes that could be selected by antiviral agents that specifically target capsid assembly.While viruses often acquire resistance to antiviral agents, resistance mutants generally exhibit lower fitness than the wildtype strain in the absence of the inhibitor (6,16,17,25) and can develop a dependency on the antiviral agent (1, 19). However, the molecular mechanism of dependency rarely, if ever, involves the productive use of the antiviral agent to elevate fitness above the uninhibited wild-type level. Many studies are conducted with animal viruses, often in clinical settings, which can impose restraints on the experimental durations. Thus, prolonged exposure to antiviral agents may be required for the emergence of a multiply mutant strain that has evolved mechanisms to productively utilize inhibitors.Due to its rapid replication, bacteriophage X174 has become an attractive model system for evolutionary studies (2,3,23,24). Selective pressures can be applied for hundreds of infection cycles in a relatively short period of time. Using the atomic structure of assembly intermediates as a guide (8, 9, 15), viral scaffolding proteins that inhibit virion assembly have been designed (4). The molecular mechanism of inhibition was characterized, and resistance mutants were isolated via onestep genetic selections (4). In this study we report the isolation of a more robust resistant mutant. The quintuple mutant was generated by propagating X174 for approximately 150 life cycles in the presence of increasing concentrations of the inhibitory protein, which was derived from the external scaffolding D protein. This protein forms asymmetric dimers that direct procapsid assembly. A...

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“…A multiple-mutant resistant strain was experimentally evolved by culturing X174 in exponential-phase cells while incrementally increasing the induction of the lethal dominant gene (12). Like other viruses that acquire resistance to antiviral agents (14,27,28,37), the resistant strain exhibits lower fitness than that of the uninhibited wild-type strain in the absence of the inhibitor. It is also dependent on the once inhibitory protein for optimal fitness, which is not uncommon (3,29).…”

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confidence: 99%

From Resistance to Stimulation: the Evolution of a Virus in the Presence of a Dominant Lethal Inhibitory Scaffolding Protein

Cherwa

Fane

2011

J Virol

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By acquiring resistance to an inhibitor, viruses can become dependent on that inhibitor for optimal fitness. However, inhibitors rarely, if ever, stimulate resistant strain fitness to values that equal or exceed the uninhibited wild-type level. This would require an adaptive mechanism that converts the inhibitor into a beneficial replication factor. Using a plasmid-encoded inhibitory external scaffolding protein that blocks X174 assembly, we previously demonstrated that such mechanisms are possible. The resistant strain, referred to as the evolved strain, contains four mutations contributing to the resistance phenotype. Three mutations confer substitutions in the coat protein, whereas the fourth mutation alters the virus-encoded external scaffolding protein. To determine whether stimulation by the inhibitory protein coevolved with resistance or whether it was acquired after resistance was firmly established, the strain temporally preceding the previously characterized mutant, referred to as the intermediary strain, was isolated and characterized. The results of the analysis indicated that the mutation in the virus-encoded external scaffolding protein was primarily responsible for stimulating strain fitness. When the mutation was placed in a wild-type background, it did not confer resistance. The mutation was also placed in cis with the plasmid-encoded dominant lethal mutation. In this configuration, the stimulating mutation exhibited no activity, regardless of the genotype (wild type, evolved, or intermediary) of the infecting virus. Thus, along with the coat protein mutations, stimulation required two external scaffolding protein genes: the once inhibitory gene and the mutant gene acquired during evolution.With developed genetics and biochemistry and solved atomic structures, bacteriophage X174 has a long and rich history as a virus assembly system (15-17, 20, 22-25). Due to its rapid replication, which allows selective pressures to be applied for hundreds of infection cycles, it recently emerged as an attractive organism for evolutionary studies (4)(5)(6)(33)(34)(35)). An evolutionary approach to assembly was used to study the evolution of resistance to a genetically engineered inhibitory protein, a dominant lethal external scaffolding protein that specifically targets procapsid morphogenesis. A multiple-mutant resistant strain was experimentally evolved by culturing X174 in exponential-phase cells while incrementally increasing the induction of the lethal dominant gene (12). Like other viruses that acquire resistance to antiviral agents (14,27,28,37), the resistant strain exhibits lower fitness than that of the uninhibited wild-type strain in the absence of the inhibitor. It is also dependent on the once inhibitory protein for optimal fitness, which is not uncommon (3, 29). But unlike similar examples, the inhibitor stimulates fitness to values equal to or above the uninhibited wild-type level, suggesting that the virus evolved a mechanism to convert the inhibitory protein into a beneficial replication factor...

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Phenotypic Characterization of Resistant Val ³⁶ Variants of Hepatitis C Virus NS3-4A Serine Protease

Cited by 90 publications

References 50 publications

Drug resistance against HCV NS3/4A inhibitors is defined by the balance of substrate recognition versus inhibitor binding

Drug resistance against HCV NS3/4A inhibitors is defined by the balance of substrate recognition versus inhibitor binding

Viral Adaptation to an Antiviral Protein Enhances the Fitness Level to Above That of the Uninhibited Wild Type

From Resistance to Stimulation: the Evolution of a Virus in the Presence of a Dominant Lethal Inhibitory Scaffolding Protein

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Phenotypic Characterization of Resistant Val 36 Variants of Hepatitis C Virus NS3-4A Serine Protease

Cited by 90 publications

References 50 publications

Drug resistance against HCV NS3/4A inhibitors is defined by the balance of substrate recognition versus inhibitor binding

Drug resistance against HCV NS3/4A inhibitors is defined by the balance of substrate recognition versus inhibitor binding

Viral Adaptation to an Antiviral Protein Enhances the Fitness Level to Above That of the Uninhibited Wild Type

From Resistance to Stimulation: the Evolution of a Virus in the Presence of a Dominant Lethal Inhibitory Scaffolding Protein

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Product

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Phenotypic Characterization of Resistant Val ³⁶ Variants of Hepatitis C Virus NS3-4A Serine Protease