Influenza neuraminidase is responsible for the escape of new viral particles from the infected cell surface. Several neuraminidase inhibitors are used clinically to treat patients or stockpiled for emergencies. However, the increasing development of viral resistance against approved inhibitors has underscored the need for the development of new antivirals effective against resistant influenza strains. A facile, sensitive, and inexpensive screening method would help achieve this goal. Recently, we described a multiwell plate-based DNA-linked inhibitor antibody assay (DIANA). This highly sensitive method can quantify femtomolar concentrations of enzymes. DIANA also has been applied to high-throughput enzyme inhibitor screening, allowing the evaluation of inhibition constants from a single inhibitor concentration. Here, we report the design, synthesis, and structural characterization of a tamiphosphor derivative linked to a reporter DNA oligonucleotide for the development of a DIANA-type assay to screen potential influenza neuraminidase inhibitors. The neuraminidase is first captured by an immobilized antibody, and the test compound competes for binding to the enzyme with the oligo-linked detection probe, which is then quantified by qPCR. We validated this novel assay by comparing it with the standard fluorometric assay and demonstrated its usefulness for sensitive neuraminidase detection as well as high-throughput screening of potential new neuraminidase inhibitors.
- MeSH
- antivirové látky chemie farmakologie MeSH
- chřipka lidská farmakoterapie enzymologie virologie MeSH
- DNA chemie MeSH
- inhibitory enzymů chemie farmakologie MeSH
- kyseliny fosforité chemie MeSH
- lidé MeSH
- neuraminidasa antagonisté a inhibitory metabolismus MeSH
- oseltamivir analogy a deriváty chemie MeSH
- preklinické hodnocení léčiv metody MeSH
- reprodukovatelnost výsledků MeSH
- virové proteiny antagonisté a inhibitory metabolismus MeSH
- virus chřipky A účinky léků enzymologie fyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Viral infection perturbs host cells and can be used to uncover regulatory mechanisms controlling cellular responses and susceptibility to infections. Using cell biological, biochemical, and genetic tools, we reveal that influenza A virus (IAV) infection induces global transcriptional defects at the 3' ends of active host genes and RNA polymerase II (RNAPII) run-through into extragenic regions. Deregulated RNAPII leads to expression of aberrant RNAs (3' extensions and host-gene fusions) that ultimately cause global transcriptional downregulation of physiological transcripts, an effect influencing antiviral response and virulence. This phenomenon occurs with multiple strains of IAV, is dependent on influenza NS1 protein, and can be modulated by SUMOylation of an intrinsically disordered region (IDR) of NS1 expressed by the 1918 pandemic IAV strain. Our data identify a strategy used by IAV to suppress host gene expression and indicate that polymorphisms in IDRs of viral proteins can affect the outcome of an infection.
- MeSH
- chřipka lidská genetika MeSH
- lidé MeSH
- RNA-polymerasa II genetika MeSH
- terminátorové oblasti (genetika) genetika MeSH
- virulence MeSH
- virus chřipky A patogenita fyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Chřipka je vysoce nakažlivé virové onemocnění, které se přenáší kapénkovou nákazou a postihuje především dýchací cesty. Téměř každý rok dochází v zimních měsících k epidemiím a v delších časových intervalech nový subtyp viru způsobí pandemii, která různě závažným průběhem postihne až 50% populace. Od roku 1997 došlo v mnoha zemích jihovýchodní Asie k rozsáhlé zhoubné epizoocii ptačí chřipky u drůbeže způsobené subtypem A H5N1, při níž onemocnělo přes 100 osob, z nichž více než polovina chorobě podlehla. Panuje obava, aby nedošlo k humanizaci uvedeného viru, který by mohl způsobit pandemii.
Influenza is a highly contagious viral disease that is transmitted by air-borne infection and involves predominantly respiratory airways. Almost every year epidemics occur in winter involving more than 50% of population to a various extent. Since 1997 several epidemics of avian influenza of a different size occurred by a subtype A H5N1 involving more than 100 patients with more than 50% mortality. There is fear about humanization of the virus that could cause a pandemic.
- MeSH
- finanční podpora výzkumu jako téma MeSH
- mutace MeSH
- virulence MeSH
- virus chřipky A fyziologie genetika imunologie MeSH
- Publikační typ
- přehledy MeSH
- MeSH
- lidé MeSH
- virus chřipky A fyziologie imunologie MeSH
- Check Tag
- lidé MeSH
- MeSH
- lidé MeSH
- neuraminidasa fyziologie MeSH
- virus chřipky A fyziologie izolace a purifikace MeSH
- Check Tag
- lidé MeSH
- MeSH
- lidé MeSH
- neuraminidasa fyziologie MeSH
- virus chřipky A fyziologie izolace a purifikace MeSH
- Check Tag
- lidé MeSH
- MeSH
- kreatinkinasa fyziologie MeSH
- lidé MeSH
- serózní membrána fyziologie virologie MeSH
- virus chřipky A fyziologie MeSH
- Check Tag
- lidé MeSH
