Generation and Characterization of Recombinant Influenza A (H1N1) Viruses Harboring Amantadine Resistance Mutations

Abed, Yacine; Goyette, Nathalie; Boivin, Guy

doi:10.1128/aac.49.2.556-559.2005

Cited by 163 publications

(138 citation statements)

References 15 publications

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“…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).…”

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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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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“…1,42 A subset of these mutations are found in infected patients following treatment with amantadine, 43 and reverseengineered viruses harboring various pore-lining mutations are able to replicate in vitro and in a mouse model. 44 However, many of these mutations give rise to somewhat attenuated viruses that are less transmissible than wild-type (WT) and tend to revert in the absence of drug pressure. 42,45 Indeed, large-scale sequencing of transmissible viruses from 1918 to 2010 showed that mutations to pore-lining residues are allowed only within the first turn of the TM helix at positions 26, 27, and 31 [ Fig.…”

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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“…It was previously reported that the serine 31 (S31) cluster is the high-affinity binding site for amantadine (42)(43)(44) and that residues S31 and valine 27 (V27) in M2 confer M2 sensitivity to amantadine (45)(46)(47)(48)(49)(50)(51)(52)(53). Wild-type MX M2 contains residues N31 and V27, while PR M2 contains N31 and threonine 27 (T27) (Fig.…”

Section: M2 Enhances Infectivity Of Ha-pseudovirusmentioning

confidence: 99%

Influenza Virus M2 Protein Ion Channel Activity Helps To Maintain Pandemic 2009 H1N1 Virus Hemagglutinin Fusion Competence during Transport to the Cell Surface

Alvarado-Facundo

Gao

Ribas‐Aparicio

et al. 2015

J Virol

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The influenza virus hemagglutinin (HA) envelope protein mediates virus entry by first binding to cell surface receptors and then fusing viral and endosomal membranes during endocytosis. Cleavage of the HA precursor (HA0) into a surface receptor-binding subunit (HA1) and a fusion-inducing transmembrane subunit (HA2) by host cell enzymes primes HA for fusion competence by repositioning the fusion peptide to the newly created N terminus of HA2. We previously reported that the influenza virus M2 protein enhances pandemic 2009 influenza A virus [(H1N1)pdm09] HA-pseudovirus infectivity, but the mechanism was unclear. In this study, using cell-cell fusion and HA-pseudovirus infectivity assays, we found that the ion channel function of M2 was required for enhancement of HA fusion and HA-pseudovirus infectivity. The M2 activity was needed only during HA biosynthesis, and proteolysis experiments indicated that M2 proton channel activity helped to protect (H1N1)pdm09 HA from premature conformational changes as it traversed low-pH compartments during transport to the cell surface. While M2 has previously been shown to protect avian influenza virus HA proteins of the H5 and H7 subtypes that have polybasic cleavage motifs, this study demonstrates that M2 can protect HA proteins from human H1N1 strains that lack a polybasic cleavage motif. This finding suggests that M2 proton channel activity may play a wider role in preserving HA fusion competence among a variety of HA subtypes, including HA proteins from emerging strains that may have reduced HA stability. IMPORTANCEInfluenza virus infects cells when the hemagglutinin (HA) surface protein undergoes irreversible pH-induced conformational changes after the virus is taken into the cell by endocytosis. HA fusion competence is primed when host cell enzymes cleave the HA precursor. The proton channel function of influenza virus M2 protein has previously been shown to protect avian influenza virus HA proteins that contain a polybasic cleavage site from pH-induced conformational changes during biosynthesis, but this effect is less well understood for human influenza virus HA proteins that lack polybasic cleavage sites. Using assays that focus on HA entry and fusion, we found that the M2 protein also protects (H1N1)pdm09 influenza A virus HA from premature conformational changes as it transits low-pH compartments during biosynthesis. This work suggests that M2 may play a wider role in preserving HA function in a variety of influenza virus subtypes that infect humans and may be especially important for HA proteins that are less stable. T he influenza virus hemagglutinin (HA) envelope protein mediates virus entry by binding to cell surface receptors, followed by endocytosis and fusion of the viral and endosomal membranes. Cellular proteases cleave the HA precursor (HA0) to generate the HA1 surface subunit, which mediates binding to cell surface sialic acid receptors, and the HA2 transmembrane subunit, which mediates membrane fusion between viral and endosomal membranes during e...

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Generation and Characterization of Recombinant Influenza A (H1N1) Viruses Harboring Amantadine Resistance Mutations

Cited by 163 publications

References 15 publications

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

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

Influenza Virus M2 Protein Ion Channel Activity Helps To Maintain Pandemic 2009 H1N1 Virus Hemagglutinin Fusion Competence during Transport to the Cell Surface

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