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Identification of critical residues of influenza neuraminidase in viral particle release.

ABSTRACT: Influenza neuraminidase (NA) is essential for virus release from its host cells and it is one of the targets for structure-based antiviral drug design.In this report, we established a pseudoviral particle release assay to study NA function, which is based on lentiviral particles pseudotyped with influenza glycoproteins HA and NA as a surrogate system. Through an extensive molecular analysis, we sought to characterize important residues governing NA function. We identified five residues of NA, 234, 241, 257, 286 and 345, four of which (except 345) map away from the active site of NA when projected onto the three-dimensional structure of avian influenza H5N1 NA, and substitutions of these residues adversely affected the NA-mediated viral particle release, suggesting that these residues are critical for NA enzymatic activity.Through extensive chimeric and mutational analyses, we have identified several residues, which map away from the active site and are critical for NA function. These findings provide new insights into NA-mediated pseudoviral particle release and may have important implications in drug design and therapeutics against influenza infection.

SUBMITTER: Tisoncik JR

PROVIDER: S-EPMC3033838 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Identification of critical residues of influenza neuraminidase in viral particle release.

Tisoncik Jennifer R JR Guo Ying Y Cordero Katie S KS Yu Jia J Wang Jianwei J Cao Youjia Y Rong Lijun L

Virology journal 20110113

<h4>Background</h4>Influenza neuraminidase (NA) is essential for virus release from its host cells and it is one of the targets for structure-based antiviral drug design.<h4>Results</h4>In this report, we established a pseudoviral particle release assay to study NA function, which is based on lentiviral particles pseudotyped with influenza glycoproteins HA and NA as a surrogate system. Through an extensive molecular analysis, we sought to characterize important residues governing NA function. We ...[more]

PMID: 21232128

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Project description:UnlabelledInfluenza A virus (IAV) attachment to and release from sialoside receptors is determined by the balance between hemagglutinin (HA) and neuraminidase (NA). The molecular determinants that mediate the specificity and activity of NA are still poorly understood. In this study, we aimed to design the optimal recombinant soluble NA protein to identify residues that affect NA enzymatic activity. To this end, recombinant soluble versions of four different NA proteins from H5N1 viruses were compared with their full-length counterparts. The soluble NA ectodomains were fused to three commonly used tetramerization domains. Our results indicate that the particular oligomerization domain used does not affect the Km value but may affect the specific enzymatic activity. This particularly holds true when the stalk domain is included and for NA ectodomains that display a low intrinsic ability to oligomerize. NA ectodomains extended with a Tetrabrachion domain, which forms a nearly parallel four-helix bundle, better mimicked the enzymatic properties of full-length proteins than when other coiled-coil tetramerization domains were used, which probably distort the stalk domain. Comparison of different NA proteins and mutagenic analysis of recombinant soluble versions thereof resulted in the identification of several residues that affected oligomerization of the NA head domain (position 95) and therefore the specific activity or sialic acid binding affinity (Km value; positions 252 and 347). This study demonstrates the potential of using recombinant soluble NA proteins to reveal determinants of NA assembly and enzymatic activity.ImportanceThe IAV HA and NA glycoproteins are important determinants of host tropism and pathogenicity. However, NA is relatively understudied compared to HA. Analysis of soluble versions of these glycoproteins is an attractive way to study their activities, as they are easily purified from cell culture media and applied in downstream assays. In the present study, we analyzed the enzymatic activity of different NA ectodomains with three commonly used tetramerization domains and compared them with full-length NA proteins. By performing a mutagenic analysis, we identified several residues that affected NA assembly, activity, and/or substrate binding. In addition, our results indicate that the design of the recombinant soluble NA protein, including the particular tetramerization domain, is an important determinant for maintaining the enzymatic properties within the head domain. NA ectodomains extended with a Tetrabrachion domain better mimicked the full-length proteins than when the other tetramerization domains were used.

Project description:Influenza A viruses possess two glycoprotein spikes on the virion surface: hemagglutinin (HA), which binds to oligosaccharides containing terminal sialic acid, and neuraminidase (NA), which removes terminal sialic acid from oligosaccharides. Hence, the interplay between these receptor-binding and receptor-destroying functions assumes major importance in viral replication. In contrast to the well-characterized role of HA in host range restriction of influenza viruses, there is only limited information on the role of NA substrate specificity in viral replication among different animal species. We therefore investigated the substrate specificities of NA for linkages between N-acetyl sialic acid and galactose (NeuAcalpha2-3Gal and NeuAcalpha2-6Gal) and for different molecular species of sialic acids (N-acetyl and N-glycolyl sialic acids) in influenza A viruses isolated from human, avian, and pig hosts. Substrate specificity assays showed that all viruses had similar specificities for NeuAcalpha2-3Gal, while the activities for NeuAcalpha2-6Gal ranged from marginal, as represented by avian and early N2 human viruses, to high (although only one-third the activity for NeuAcalpha2-3Gal), as represented by swine and more recent N2 human viruses. Using site-specific mutagenesis, we identified in the earliest human virus with a detectable increase in NeuAcalpha2-6Gal specificity a change at position 275 (from isoleucine to valine) that enhanced the specificity for this substrate. Valine at position 275 was maintained in all later human viruses as well as swine viruses. A similar examination of N-glycolylneuraminic acid (NeuGc) specificity showed that avian viruses and most human viruses had low to moderate activity for this substrate, with the exception of most human viruses isolated between 1967 and 1969, whose NeuGc specificity was as high as that of swine viruses. The amino acid at position 431 was found to determine the level of NeuGc specificity of NA: lysine conferred high NeuGc specificity, while proline, glutamine, and glutamic acid were associated with lower NeuGc specificity. Both residues 275 and 431 lie close to the enzymatic active site but are not directly involved in the reaction mechanism. This finding suggests that the adaptation of NA to different substrates occurs by a mechanism of amino acid substitutions that subtly alter the conformation of NA in and around the active site to facilitate the binding of different species of sialic acid.

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Identification of critical residues of influenza neuraminidase in viral particle release.

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Identification of critical residues of influenza neuraminidase in viral particle release.

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