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New details of HCV NS3/4A proteinase functionality revealed by a high-throughput cleavage assay.

ABSTRACT: The hepatitis C virus (HCV) genome encodes a long polyprotein, which is processed by host cell and viral proteases to the individual structural and non-structural (NS) proteins. HCV NS3/4A serine proteinase (NS3/4A) is a non-covalent heterodimer of the N-terminal, ?180-residue portion of the 631-residue NS3 protein with the NS4A co-factor. NS3/4A cleaves the polyprotein sequence at four specific regions. NS3/4A is essential for viral replication and has been considered an attractive drug target.Using a novel multiplex cleavage assay and over 2,660 peptide sequences derived from the polyprotein and from introducing mutations into the known NS3/4A cleavage sites, we obtained the first detailed fingerprint of NS3/4A cleavage preferences. Our data identified structural requirements illuminating the importance of both the short-range (P1-P1') and long-range (P6-P5) interactions in defining the NS3/4A substrate cleavage specificity. A newly observed feature of NS3/4A was a high frequency of either Asp or Glu at both P5 and P6 positions in a subset of the most efficient NS3/4A substrates. In turn, aberrations of this negatively charged sequence such as an insertion of a positively charged or hydrophobic residue between the negatively charged residues resulted in inefficient substrates. Because NS5B misincorporates bases at a high rate, HCV constantly mutates as it replicates. Our analysis revealed that mutations do not interfere with polyprotein processing in over 5,000 HCV isolates indicating a pivotal role of NS3/4A proteolysis in the virus life cycle.Our multiplex assay technology in light of the growing appreciation of the role of proteolytic processes in human health and disease will likely have widespread applications in the proteolysis research field and provide new therapeutic opportunities.

SUBMITTER: Shiryaev SA

PROVIDER: S-EPMC3338790 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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New details of HCV NS3/4A proteinase functionality revealed by a high-throughput cleavage assay.

Shiryaev Sergey A SA Thomsen Elliot R ER Cieplak Piotr P Chudin Eugene E Cheltsov Anton V AV Chee Mark S MS Kozlov Igor A IA Strongin Alex Y AY

PloS one 20120427 4

<h4>Background</h4>The hepatitis C virus (HCV) genome encodes a long polyprotein, which is processed by host cell and viral proteases to the individual structural and non-structural (NS) proteins. HCV NS3/4A serine proteinase (NS3/4A) is a non-covalent heterodimer of the N-terminal, ∼180-residue portion of the 631-residue NS3 protein with the NS4A co-factor. NS3/4A cleaves the polyprotein sequence at four specific regions. NS3/4A is essential for viral replication and has been considered an attr ...[more]

PMID: 22558217

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Project description:BackgroundHepatitis C is a treatment-resistant disease affecting millions of people worldwide. The hepatitis C virus (HCV) genome is a single-stranded RNA molecule. After infection of the host cell, viral RNA is translated into a polyprotein that is cleaved by host and viral proteinases into functional, structural and non-structural, viral proteins. Cleavage of the polyprotein involves the viral NS3/4A proteinase, a proven drug target. HCV mutates as it replicates and, as a result, multiple emerging quasispecies become rapidly resistant to anti-virals, including NS3/4A inhibitors.Methodology/principal findingsTo circumvent drug resistance and complement the existing anti-virals, NS3/4A inhibitors, which are additional and distinct from the FDA-approved telaprevir and boceprevir α-ketoamide inhibitors, are required. To test potential new avenues for inhibitor development, we have probed several distinct exosites of NS3/4A which are either outside of or partially overlapping with the active site groove of the proteinase. For this purpose, we employed virtual ligand screening using the 275,000 compound library of the Developmental Therapeutics Program (NCI/NIH) and the X-ray crystal structure of NS3/4A as a ligand source and a target, respectively. As a result, we identified several novel, previously uncharacterized, nanomolar range inhibitory scaffolds, which suppressed of the NS3/4A activity in vitro and replication of a sub-genomic HCV RNA replicon with a luciferase reporter in human hepatocarcinoma cells. The binding sites of these novel inhibitors do not significantly overlap with those of α-ketoamides. As a result, the most common resistant mutations, including V36M, R155K, A156T, D168A and V170A, did not considerably diminish the inhibitory potency of certain novel inhibitor scaffolds we identified.Conclusions/significanceOverall, the further optimization of both the in silico strategy and software platform we developed and lead compounds we identified may lead to advances in novel anti-virals.

Project description:We have investigated the ability of hepatitis C virus non-structural (NS) 3/4A-DNA-based vaccines to activate long-term cell-mediated immune responses in mice. Wild-type and synthetic codon optimized (co) NS3/4A DNA vaccines have previously been shown to be immunogenic in mice, rabbits and humans, although we have very poor knowledge about the longevity of the immune responses primed. We therefore analyzed the functionality of primed NS3/4A-specific immune responses in BALB/c (H-2(d)) and/or C57BL/6J (H-2(b)) mice 1, 2, 3, 4, 6, 12 and 16 months after the last immunization. Mice were immunized one, two, three or four times using gene gun delivery to the skin or by intramuscular administration. Immunological responses after immunization were monitored by protection against in vivo challenge of NS3/4A-expressing syngeneic tumor cells. In addition, functionality of the NS3/4A-specific T cells was analyzed by a standard cytotoxicity assay. First, we identified a new unique murine H-2(d)-restricted NS3/4A cytotoxic T lymphocyte (CTL) epitope, which enabled us to study the epitope-specific immune responses. Our results show that the coNS3/4A vaccine was highly immunogenic by determination of interferon-?/tumor necrosis factor-? production and lytic cytotoxic T cells, which could efficiently inhibit in vivo tumor growth. Importantly, we showed that one to four monthly immunizations protected mice from tumor development when challenged up to 16 months after the last immunization. When determining the functionality of NS3/4A-specific T cells in vitro, we showed detectable lytic activity up to 12 months after the last immunization. Thus, NS3/4A-based DNA vaccines activate potent cellular immune responses that are present and function in both BALB/c and C57BL/6J mice up to 12-16 months after the last immunization. The induction of long-term immunity after NS3/4A DNA immunization has not been shown previously and supports the use of NS3/4A in hepatitis C virus vaccine compositions.

Dataset Information

New details of HCV NS3/4A proteinase functionality revealed by a high-throughput cleavage assay.

Publications

New details of HCV NS3/4A proteinase functionality revealed by a high-throughput cleavage assay.

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