Genomic events underlying the changes in adamantane resistance among influenza A(H3N2) viruses during 2006–2008

Deyde, Varough; Garten, Rebecca; Sheu, Tiffany G.; Smith, Catherine; Myrick, Allison; Barnes, John; Xu, Xinhua; Shaw, Michael; Klimov, Alexander; Gubareva, Larisa V.

doi:10.1111/j.1750-2659.2009.00103.x

Cited by 21 publications

(18 citation statements)

References 41 publications

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“…Analysis of 95 H3N2 viruses collected during the following influenza season (2008-2009), using the RT-PCR/ESI-MS assay, revealed that they all retained the genomic prints CCDFAA or CCLFAA, which are characteristic of the D lineage, with no apparent evidence of major genetic changes based on this assay. These observations were consistent with the results obtained based on phylogenetic analyses [24].…”

Section: Rt-pcr/esi-ms Approach In Detection and Characterization Of Insupporting

confidence: 93%

“…in prior seasons, specifically the N-lineage [12,20]. Comparison of the RT-PCR/ESI-MS results with full-genome sequencing and phylogenetic data of the same 65 viruses showed that the 20 representative genomic prints identified clustered according to the four genetic lineages (A-D) determined by phylogenetic ana lysis [24].…”

Section: Rt-pcr/esi-ms Approach In Detection and Characterization Of Inmentioning

confidence: 95%

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RT-PCR/electrospray ionization mass spectrometry approach in detection and characterization of influenza viruses

Deyde

Sampath²,

Gubareva

2011

Expert Review of Molecular Diagnostics

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Reverse-transcription PCR (RT-PCR) coupled with electrospray ionization mass spectrometry (ESI-MS) is a high-throughput nucleic acid-based technology that relies on the accurate measurement of the molecular weight of PCR amplicons that can be used to deduce the base counts (number of As, Gs, Cs and Ts) of DNA. These amplicons represent highly variable regions with information-rich sequences, which are flanked by broad-range primers designed based on highly conserved loci. This technology was first introduced in 2005 for microbial identification and subtyping, and was later applied to influenza virus detection and identification. The influenza RT-PCR/ESI-MS assay allows analysis of approximately 300 samples per 24 h, and aids in the characterization of influenza viruses based on their 'core' gene signatures. Notably, this assay was used to identify one of the first cases of the 2009 H1N1 pandemic viruses. One of the main advantages of the RT-PCR/ESI-MS technology is its universality and adaptability for pathogen characterization. Efforts are being made to customize the currently used influenza surveillance assay for use in the diagnosis of the H1N1 pandemic virus. In this article, we provide a summary of known applications of the RT-PCR/ESI-MS assay in the field of influenza.

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Section: Rt-pcr/esi-ms Approach In Detection and Characterization Of Insupporting

confidence: 93%

Section: Rt-pcr/esi-ms Approach In Detection and Characterization Of Inmentioning

confidence: 95%

RT-PCR/electrospray ionization mass spectrometry approach in detection and characterization of influenza viruses

Deyde

Sampath²,

Gubareva

2011

Expert Review of Molecular Diagnostics

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“…Indeed, large-scale sequencing of transmissible viruses isolated as early as 1918 showed that mutations to pore-lining residues are allowed only within the first turn of the transmembrane (TM) helix at positions 26, 27, and 31 (10). S31N has long been the predominant amantadine-resistant mutation in M2 (11)(12)(13)(14). It predominated in 98-100% of the transmissible amantadineresistant H1N1, H5N1, and H3N2 strains isolated from humans, birds, and swine in the past decade.…”

Section: M2-s31n Inhibitormentioning

confidence: 99%

Structure and inhibition of the drug-resistant S31N mutant of the M2 ion channel of influenza A virus

Wang

Ma³

et al. 2013

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

204

350

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The influenza A virus M2 proton channel (A/M2) is the target of the antiviral drugs amantadine and rimantadine, whose use has been discontinued due to widespread drug resistance. Among the handful of drug-resistant mutants, S31N is found in more than 95% of the currently circulating viruses and shows greatly decreased inhibition by amantadine. The discovery of inhibitors of S31N has been hampered by the limited size, polarity, and dynamic nature of its amantadine-binding site. Nevertheless, we have discovered smallmolecule drugs that inhibit S31N with potencies greater than amantadine's potency against WT M2. Drug binding locks the protein into a well-defined conformation, and the NMR structure of the complex shows the drug bound in the homotetrameric channel, threaded between the side chains of Asn31. Unrestrained molecular dynamics simulations predicted the same binding site. This S31N inhibitor, like other potent M2 inhibitors, contains a charged ammonium group. The ammonium binds as a hydrate to one of three sites aligned along the central cavity that appear to be hotspots for inhibition. These sites might stabilize hydronium-like species formed as protons diffuse through the outer channel to the proton-shuttling residue His37 near the cytoplasmic end of the channel.M2-S31N mutant structure | membrane protein structure | M2-S31N inhibitorT he influenza A virus M2 proton channel (A/M2) is the target of the antiviral drugs amantadine and rimantadine (1-3), which bind directly to the pore of the channel (2-4). Although amantadine has been widely used for several decades, drug resistance has curtailed the use of this family of drugs. Many amantadineresistant influenza viruses can be selected in cell culture (5, 6). A subset of these mutations is found in infected patients undergoing treatment with amantadine (7), and reverse-engineered viruses harboring various pore-lining mutations are competent to replicate in the mouse (8). However, many of these mutations give rise to somewhat attenuated viruses that are less transmissible than WT virus, and they tend to revert in the absence of drug pressure (6, 9). Indeed, large-scale sequencing of transmissible viruses isolated as early as 1918 showed that mutations to pore-lining residues are allowed only within the first turn of the transmembrane (TM) helix at positions 26, 27, and 31 (10). S31N has long been the predominant amantadine-resistant mutation in M2 (11)(12)(13)(14). It predominated in 98-100% of the transmissible amantadineresistant H1N1, H5N1, and H3N2 strains isolated from humans, birds, and swine in the past decade. V27A and L26F are less frequent mutations (10,11,15). Extensive studies of point mutations to the pore-lining residues of M2 have been conducted to understand the paucity of natural variants (16,17). Numerous mutants in the N-terminal aqueous pore retained the ability to conduct protons selectively over other ions, although the magnitude and pH dependence of their conduction varied. However, only a few mutations at the most distal sites, V27A,...

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“…1(A)]. S31N has long been the dominant amantadine-resistant mutation in M2, [46][47][48][49] accounting for 98-100%…”

Section: Natural and Artificial Sequence Variants Of M2mentioning

confidence: 99%

Structural basis for proton conduction and inhibition by the influenza M2 protein

2012

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The influenza M2 protein forms an acid-activated and drug-sensitive proton channel in the virus envelope that is important for the virus lifecycle. The functional properties and high-resolution structures of this proton channel have been extensively studied to understand the mechanisms of proton conduction and drug inhibition. We review biochemical and electrophysiological studies of M2 and discuss how high-resolution structures have transformed our understanding of this proton channel. Comparison of structures obtained in different membrane-mimetic solvents and under different pH using X-ray crystallography, solution NMR, and solid-state NMR spectroscopy revealed how the M2 structure depends on the environment and showed that the pharmacologically relevant drug-binding site lies in the transmembrane (TM) pore. Competing models of proton conduction have been evaluated using biochemical experiments, high-resolution structural methods, and computational modeling. These results are converging to a model in which a histidine residue in the TM domain mediates proton relay with water, aided by microsecond conformational dynamics of the imidazole ring. These mechanistic insights are guiding the design of new inhibitors that target drug-resistant M2 variants and may be relevant for other proton channels.

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Genomic events underlying the changes in adamantane resistance among influenza A(H3N2) viruses during 2006–2008

Cited by 21 publications

References 41 publications

RT-PCR/electrospray ionization mass spectrometry approach in detection and characterization of influenza viruses

RT-PCR/electrospray ionization mass spectrometry approach in detection and characterization of influenza viruses

Structure and inhibition of the drug-resistant S31N mutant of the M2 ion channel of influenza A virus

Structural basis for proton conduction and inhibition by the influenza M2 protein

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