Influenza neuraminidase

Air, Gillian M.

doi:10.1111/j.1750-2659.2011.00304.x

Cited by 208 publications

(220 citation statements)

References 102 publications

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“…However, the structural and functional roles of the stalk domain of NA were unknown thus far, and there was no report about the glycosylation of these canonical glycosites (19)(20)(21)(22)(23). In this study, we prove that the glycosites in the stalk domain of NA are glycosylated to regulate the activity, affinity, and specificity of NA to modulate IAV replication, suggesting that the glycans in the stalk domain of NA play an important role in the virulence and transmission of IAV.…”

Section: Discussionmentioning

confidence: 51%

Influenza A surface glycosylation and vaccine design

Lin

Tsai

et al. 2016

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

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We have shown that glycosylation of influenza A virus (IAV) hemagglutinin (HA), especially at position N-27, is crucial for HA folding and virus survival. However, it is not known whether the glycosylation of HA and the other two major IAV surface glycoproteins, neuraminidase (NA) and M2 ion channel, is essential for the replication of IAV. Here, we show that glycosylation of HA at N-142 modulates virus infectivity and host immune response. Glycosylation of NA in the stalk region affects its structure, activity, and specificity, thereby modulating virus release and virulence, and glycosylation at the catalytic domain affects its thermostability; however, glycosylation of M2 had no effect on its function. In addition, using IAV without the stalk and catalytic domains of NA as a live attenuated vaccine was shown to confer a strong IAVspecific CD8+ T-cell response and a strong cross-strain as well as cross-subtype protection against various virus strains.influenza A virus | glycosylation | vaccine design I nfluenza A viruses (IAV) belong to the Orthomyxoviridae family and can circulate widely and cross interspecies barriers through the highly antigenic drift and shift of the 18 subtypes of HA and 11 subtypes of neuraminidase (NA) (1, 2). In addition, the posttranslational modification of the IAV surface proteins is important to circumvent host defense and support the virus life cycle (3).HA is a major surface glycoprotein of IAV and is involved in viral infection via binding to sialic acid (SA)-containing glycans on the surface of host cell (3). The other major glycoprotein of IAV, NA, is involved in the cleavage of SA on the host cell receptor to facilitate the release of viral particles to infect other cells (4). M2, the third surface protein of IAV, has ion channel activity to regulate virus penetration and uncoating (1). All surface proteins interact with M1 protein for virus assembly and release (5).The modification of HA and NA by N-glycosylation is important in the IAV life cycle (1, 6-8). Previously, we have shown that the glycosylation of HA affects its receptor binding, immune response, and structural stability, and glycosite 27 is essential for retaining the structural integrity of HA and its receptor binding (6, 9). In addition, using the monoglycosylated HA as immunogen, it showed a broader protection against various IAV subtypes compared with the fully glycosylated version (10). However, it is not clear whether the other specific glycosites and their glycan structures on HA regulate its functions. With regard to NA, the functional roles of its glycosylation are not well understood, though N-glycosylation was reported to stabilize the protein from protease digestion and may affect the enzyme activity (11,12). The aim of this study was to understand the functional effects of glycosylation on HA, NA, and M2, and how glycosylation of the surface proteins affects the life cycle of IAV. ResultsGlycosylation of HA at N-142. Because several glycosylation sites (glycosites 27, 40, 176, 303, and 497) on HA are ...

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Section: Discussionmentioning

confidence: 51%

Influenza A surface glycosylation and vaccine design

Lin

Tsai

et al. 2016

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

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“…5). Although the GCN4-extended HN and VN proteins differed dramatically in their K m values and in their specific activity, none of these residues belong to the active site or framework residues (16)(17)(18) (Fig. 5 and 6).…”

Section: Construction and Expression Of Recombinant Na Proteinsmentioning

confidence: 99%

“…Each blade is formed by four anti-parallel beta sheets, which are stabilized by disulfide bonds and connected by loops of various lengths. The active site of NA is composed of highly conserved catalytic and structural residues that either directly contact the SIA substrate or hold the catalytic residues in place (16)(17)(18). Tetramerization of the NA protein is important for the formation of the active site and synthesis of active enzymes (19).…”

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confidence: 99%

Identification of Residues That Affect Oligomerization and/or Enzymatic Activity of Influenza Virus H5N1 Neuraminidase Proteins

Dai

Guo

Dortmans

et al. 2016

J Virol

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Influenza 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 K m 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 (K m 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 fulllength 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. V irus particles contain dedicated proteins that recognize cell surface molecules. While some viruses evolved to bind specific protein receptors, others bind carbohydrate moieties, such as sialic acids (SIAs), that are omnipresent on the cell surface as well as in the mucus. Several of the latter viruses, including influenza A virus (IAV), carry receptor-destroying activities in addit...

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“…1A). The RNA-tag was inserted into the region of the NA gene that encodes the NA stalk (12). Analysis of wild-type and recombinant viruses revealed that virus growth was unaffected by the presence of the RNA tag ( Fig.…”

Section: Significancementioning

confidence: 99%

Isolation and characterization of the positive-sense replicative intermediate of a negative-strand RNA virus

York

Hengrung

Vreede

et al. 2013

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

116

129

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Negative-strand RNA viruses represent a significant class of important pathogens that cause substantial morbidity and mortality in human and animal hosts worldwide. A defining feature of these viruses is that their single-stranded RNA genomes are of opposite polarity to messenger RNA and are replicated through a positivesense intermediate. The replicative intermediate is thought to exist as a complementary ribonucleoprotein (cRNP) complex. However, isolation of such complexes from infected cells has never been accomplished. Here we report the development of an RNA-based affinity-purification strategy for the isolation of cRNPs of influenza A virus from infected cells. This technological advance enabled the structural and functional characterization of this elusive but essential component of the viral RNA replication machine. The cRNP exhibits a filamentous double-helical organization with defined termini, containing the viral RNA-dependent RNA polymerase (RdRp) at one end and a loop structure at the other end. In vitro characterization of cRNP activity yielded mechanistic insights into the workings of this RNA synthesis machine. In particular, we found that cRNPs show activity in vitro only in the presence of added RdRp. Intriguingly, a replication-inactive RdRp mutant was also able to activate cRNP-templated viral RNA synthesis. We propose a model of influenza virus genome replication that relies on the trans-activation of the cRNP-associated RdRp. The described purification strategy should be applicable to other negative-strand RNA viruses and will promote studies into their replication mechanisms.

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Influenza neuraminidase

Cited by 208 publications

References 102 publications

Influenza A surface glycosylation and vaccine design

Influenza A surface glycosylation and vaccine design

Identification of Residues That Affect Oligomerization and/or Enzymatic Activity of Influenza Virus H5N1 Neuraminidase Proteins

Isolation and characterization of the positive-sense replicative intermediate of a negative-strand RNA virus

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