Site‐directed mutagenesis of catalytic residues of influenza virus neuraminidase as an aid to drug design

Ghate, Anita A.; Air, Gillian

doi:10.1046/j.1432-1327.1998.2580320.x

Cited by 51 publications

(48 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Except for the much weaker activities, the mutant NA shows substrate specificities toward ␣2-3 sialosides that are similar to those of the wild-type NA. The major differences in cleavage activities of the wild-type and mutant NAs indicate that D151 is indeed essential for optimal catalysis, consistent with previous structural and mechanistic studies that D151 plays a critical role in stabilizing the transition state intermediate in the catalytic reaction (3,8,11).…”

Section: Resultssupporting

confidence: 87%

Influenza Virus Neuraminidases with Reduced Enzymatic Activity That Avidly Bind Sialic Acid Receptors

Zhu

McBride

Nycholat

et al. 2012

J Virol

128

123

View full text Add to dashboard Cite

Influenza virus neuraminidase (NA) cleaves off sialic acid from cellular receptors of hemagglutinin (HA) to enable progeny escape from infected cells. However, NA variants (D151G) of recent human H3N2 viruses have also been reported to bind receptors on red blood cells, but the nature of these receptors and the effect of the mutation on NA activity were not established. Here, we compare the functional and structural properties of a human H3N2 NA from A/Tanzania/205/2010 and its D151G mutant, which supports HA-independent receptor binding. While the wild-type NA efficiently cleaves sialic acid from both ␣2-6-and ␣2-3-linked glycans, the mutant exhibits much reduced enzymatic activity toward both types of sialosides. Conversely, while wild-type NA shows no detectable binding to sialosides, the D151G NA exhibits avid binding with broad specificity toward ␣2-3 sialosides. D151G NA binds the 3= sialyllactosamine (3=-SLN) and 6=-SLN sialosides with equilibrium dissociation constant (K D ) values of 30.0 M and 645 M, respectively, which correspond to much higher affinities than the corresponding affinities (low mM) of HA to these glycans. Crystal structures of wild-type and mutant NAs reveal the structural basis for glycan binding in the active site by exclusively impairing the glycosidic bond hydrolysis step. The general significance of D151 among influenza virus NAs was further explored by introducing the D151G mutation into three N1 NAs and one N2 NA, which all exhibited reduced enzymatic activity and preferential binding to ␣2-3 sialosides. Since the enzymatic and binding activities of NAs are not routinely assessed, the potential for NA receptor binding to contribute to influenza virus biology may be underappreciated.

show abstract

Section: Resultssupporting

confidence: 87%

Influenza Virus Neuraminidases with Reduced Enzymatic Activity That Avidly Bind Sialic Acid Receptors

Zhu

McBride

Nycholat

et al. 2012

J Virol

128

123

View full text Add to dashboard Cite

show abstract

“…Although Ghate et al 22 were successful with the analysis of Tyr406Phe NA from influenza B 22 , a previous report by Lentz et al 25 demonstrated that the phenylalanine 406 substitution actually results in an aberrantly processed influenza A N2 with loss of NA activity. Therefore, we chose a different approach by using a baculovirus expression system to produce recombinant N2 (rN2) from pandemic A/RI/5 þ /1957(H2N2) with a Tyr406Asp substitution.…”

Section: Resultsmentioning

confidence: 99%

“…Leaving group effect and kinetic isotope effect experiments with influenza NA carried out by Guo et al 16 and Chou et al 20 are compatible with the classical mechanism that includes a covalent enzyme-sialosyl intermediate. Studies by Burmeister et al 21 and Ghate et al 22 also suggest that Tyr406 has an important role in the influenza NA mechanism. However, numerous studies still indicate that the influenza NA may possibly function in a unique manner.…”

mentioning

confidence: 93%

Influenza neuraminidase operates via a nucleophilic mechanism and can be targeted by covalent inhibitors

et al. 2013

View full text Add to dashboard Cite

“…The potential ability of other conserved and/or semiconserved NA residues to confer resistance to NAIs requires further exploration, and additional information about the biological properties of the conserved NA residues will also help to refine the design of existing NAIs. Mutagenesis studies of the conserved NA residues using expressed NA proteins of A/Tokyo/3/67 (H2N2) and B/Lee/40 viruses showed that amino acid substitutions at conserved NA residues may decrease NA enzymatic activity (10,22,23,37). As an aid to drug design, site-directed mutagenesis has been used to investigate the effect of mutation of conserved NA residues of B/Lee/40; however, the NAI sensitivity of the expressed NA proteins was not assayed (10).…”

mentioning

confidence: 99%

“…Mutagenesis studies of the conserved NA residues using expressed NA proteins of A/Tokyo/3/67 (H2N2) and B/Lee/40 viruses showed that amino acid substitutions at conserved NA residues may decrease NA enzymatic activity (10,22,23,37). As an aid to drug design, site-directed mutagenesis has been used to investigate the effect of mutation of conserved NA residues of B/Lee/40; however, the NAI sensitivity of the expressed NA proteins was not assayed (10). Amino acid substitution of residues 119 and 227 in the N9 glycoprotein of NWS-G70c virus can decrease NA enzymatic activity, and expressed NA with E119Q/T/G/A/V mutations showed reduced sensitivity to 4-guanidino-Neu5Ac2en (zanamivir) (11).…”

mentioning

confidence: 99%

Importance of Neuraminidase Active-Site Residues to the Neuraminidase Inhibitor Resistance of Influenza Viruses

Yen

Hoffmann

Taylor

et al. 2006

J Virol

173

135

View full text Add to dashboard Cite

Neuraminidase inhibitors (NAIs) are antivirals designed to target conserved residues at the neuraminidase (NA) enzyme active site in influenza A and B viruses. The conserved residues that interact with NAIs are under selective pressure, but only a few have been linked to resistance. In the A/Wuhan/359/95 (H3N2) recombinant virus background, we characterized seven charged, conserved NA residues (R118, R371, E227, R152, R224, E276, and D151) that directly interact with the NAIs but have not been reported to confer resistance to NAIs. These NA residues were replaced with amino acids that possess side chains having similar properties to maintain their original charge. The NA mutations we introduced significantly decreased NA activity compared to that of the A/Wuhan/359/95 recombinant wild-type and R292K (an NA mutation frequently reported to confer resistance) viruses, which were analyzed for comparison. However, the recombinant viruses differed in replication efficiency when we serially passaged them in vitro; the growth of the R118K and E227D viruses was most impaired. The R224K, E276D, and R371K mutations conferred resistance to both zanamivir and oseltamivir, while the D151E mutation reduced susceptibility to oseltamivir only (ϳ10-fold) and the R152K mutation did not alter susceptibility to either drug. Because the R224K mutation was genetically unstable and the emergence of the R371K mutation in the N2 subtype is statistically unlikely, our results suggest that only the E276D mutation is likely to emerge under selective pressure. The results of our study may help to optimize the design of NAIs.Analysis of the influenza virus neuraminidase (NA) active site revealed residues that are conserved in all NA subtypes (6), including catalytic sites (R118, D151, R152, R224, E276, R292, R371, and Y406) (in N2 numbering) that directly interact with the substrate and framework sites (E119, R156, W178, S179, D/N198, I222, E227, H274, E277, N294, and E425) that support the catalytic residues (3,7,8,16). The design of NA inhibitors (NAIs) was based on the conserved structure of the NA active site (36). NAIs interrupt the virus replication cycle by preventing the release of virus from infected cells and may interfere with the initiation of infection (6, 24).The optimal design of an antimicrobial compound requires an understanding of the molecular mechanisms that confer resistance to that agent. NAIs interact with multiple residues in the NA active site, but NA mutations selected from in vitro or in vivo experiments are limited to several conserved or semiconserved residues: R292K and E119G/A/D/V in N9 and N2 subtypes; H274Y in the N1 subtype; and E119G, D198N, R152K in influenza B virus NA (12,26,27). Influenza virus variants with the N294S mutation in NA were recently isolated after oseltamivir treatment from patients infected with either H3N2 or H5N1 influenza viruses (20,21). In addition, broad screening for the susceptibility of influenza virus to NAIs also identified viruses with natural variations at several conserved or...

show abstract

Site‐directed mutagenesis of catalytic residues of influenza virus neuraminidase as an aid to drug design

Cited by 51 publications

References 43 publications

Influenza Virus Neuraminidases with Reduced Enzymatic Activity That Avidly Bind Sialic Acid Receptors

Influenza Virus Neuraminidases with Reduced Enzymatic Activity That Avidly Bind Sialic Acid Receptors

Influenza neuraminidase operates via a nucleophilic mechanism and can be targeted by covalent inhibitors

Importance of Neuraminidase Active-Site Residues to the Neuraminidase Inhibitor Resistance of Influenza Viruses

Contact Info

Product

Resources

About