Amantadine Resistance among Porcine H1N1, H1N2, and H3N2 Influenza A Viruses Isolated in Germany between 1981 and 2001

Schmidtke, Michaela; Zell, Roland; Bauer, K.; Krumbholz, Andi; Schrader, Christina; Suess, Jochen; Wutzler, Peter

doi:10.1159/000094244

Cited by 43 publications

(36 citation statements)

References 45 publications

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“…The influenza viruses of various hosts acquired the mutation at position 31 independently and almost simultaneously. Schmidtke et al reported the emergence of amantadine-resistant strains of swine influenza virus in the 1980s and suggested that this might have been caused by a reassortment event and that further reassortment between these swine and human influenza viruses could cause an increase in the amantadineresistant M gene in human influenza virus (32). Our results are contrary to this suggestion.…”

Section: Discussioncontrasting

confidence: 99%

Large-Scale Sequence Analysis of M Gene of Influenza A Viruses from Different Species: Mechanisms for Emergence and Spread of Amantadine Resistance

Furuse

Suzuki

Oshitani

2009

Antimicrob Agents Chemother

114

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Influenza A virus infects many species, and amantadine is used as an antiviral agent. Recently, a substantial increase in amantadine-resistant strains has been reported, most of which have a substitution at amino acid position 31 in the M2 gene. Understanding the mechanism responsible for the emergence and spread of antiviral resistance is important for developing a treatment protocol for seasonal influenza and for deciding on a policy for antiviral stockpiling for pandemic influenza. The present study was conducted to identify the existence of drug pressure on the emergence and spread of amantadine-resistant influenza A viruses. We analyzed data on more than 5,000 virus sequences and constructed a phylogenetic tree to calculate selective pressures on sites in the M2 gene associated with amantadine resistance (positions 26, 27, 30, and 31) among different hosts. The phylogenetic tree revealed that the emergence and spread of the drug-resistant M gene in different hosts and subtypes were independent and not through reassortment. For human influenza virus, positive selection was detected only at position 27. Selective pressures on the sites were not always higher for human influenza virus than for viruses of other hosts. Additionally, selective pressure on position 31 did not increase after the introduction of amantadine. Although there is a possibility of drug pressure on human influenza virus, we could not find positive pressure on position 31. Because the recent rapid increase in drug-resistant virus is associated with the substitution at position 31, the resistance may not be related to drug use.

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Section: Discussioncontrasting

confidence: 99%

Large-Scale Sequence Analysis of M Gene of Influenza A Viruses from Different Species: Mechanisms for Emergence and Spread of Amantadine Resistance

Furuse

Suzuki

Oshitani

2009

Antimicrob Agents Chemother

114

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“…The NMR structure of the complex of M2WJ332 with the channel was solved for a peptide spanning from 19 to 49, which includes the TM and portions of the cytoplasmic domain and ectodomain. Two-dimensional 15 N heteronuclear single quantum coherence (HSQC) spectra for this peptide showed multiple sets of peaks of weak intensity, indicative of multiple conformations. On addition of excess drug, the total intensity increased and only one set of peaks per subunit was observed ( Fig.…”

Section: M2-s31n Inhibitormentioning

confidence: 98%

“…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).…”

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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“…11,50-60 V27A and L26F are less frequent and generally have been found in nonpandemic amantadine-resistant H1N1. 48,61,62 Extensive studies of point mutations to the pore-lining residues of M2 have been carried out to probe the conductance mechanism and to identify additional sites that might impart amantadine-resistance.…”

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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Amantadine Resistance among Porcine H1N1, H1N2, and H3N2 Influenza A Viruses Isolated in Germany between 1981 and 2001

Cited by 43 publications

References 45 publications

Large-Scale Sequence Analysis of M Gene of Influenza A Viruses from Different Species: Mechanisms for Emergence and Spread of Amantadine Resistance

Large-Scale Sequence Analysis of M Gene of Influenza A Viruses from Different Species: Mechanisms for Emergence and Spread of Amantadine Resistance

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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