Assembly of Subtype 1 Influenza Neuraminidase Is Driven by Both the Transmembrane and Head Domains

Silva, Diogo V. da; Nordholm, Johan; Madjo, Ursula; Pfeiffer, Annika; Daniels, Robert

doi:10.1074/jbc.m112.424150

Cited by 45 publications

(63 citation statements)

References 49 publications

(46 reference statements)

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“…This domain forms a nearly parallel four-helix bundle which probably better mimics oligomerization driven by the TMD and which suffices to overcome the oligomerization defect in the VN head domain. These results are in agreement with previous studies that showed the importance of the TMD for folding and assembly of the NA head domain (20,21).…”

Section: Discussionsupporting

confidence: 83%

“…To test this hypothesis, we expressed the HN and VN head domains in the absence of the artificial tetramerization domain. In addition, the stalk domain was deleted as it was previously shown that removal of the NA stalk domain restores NA activity in the absence of the TMD and an artificial tetramerization domain (20). Removal of the stalk domain from the GCN4-pLI-extended NA proteins (GCN4-NA head ) had a small negative effect for the HN protein but clearly increased the activity of the VN protein (Fig.…”

Section: Construction and Expression Of Recombinant Na Proteinsmentioning

confidence: 99%

“…Tetramerization of the NA protein is important for the formation of the active site and synthesis of active enzymes (19). Recent studies indicate that the neuraminidase TMD facilitates oligomerization of NA in coordination with the head domain to reach optimal assembly (20,21).…”

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confidence: 99%

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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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Section: Discussionsupporting

confidence: 83%

Section: Construction and Expression Of Recombinant Na Proteinsmentioning

confidence: 99%

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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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“…1A [a and e are the polar faces of the TMD model]) (20,21). Contradictory to the need to conserve hydrophobicity for membrane integration, our analysis of the N1 TMDs in human IAVs showed that they have become increasingly more polar since 1918, which results in a stronger TMD association (21).…”

mentioning

confidence: 99%

“…In this construct, the a face Asn residues at positions 21 and 28 in the pN1 TMD were mutated to Ala to produce the pN1 TMD⌬A chimera. For individual analysis, full-length NA (from WSN) and the pN1 TMD and pN1 TMD⌬A chimeras were fused to the C-terminal Myc-His tag in the mammalian expression vector pcDNA3.1A-Myc-His (Invitrogen) by PCR overlap cloning as previously described (20). The pBLM plasmids, used for TMD interaction studies, containing the NA TMDs are previously described (21).…”

Section: Plasmidsmentioning

confidence: 99%

The Influenza Virus Neuraminidase Protein Transmembrane and Head Domains Have Coevolved

Silva

Nordholm

Dou

et al. 2015

J Virol

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Transmembrane domains (TMDs) from single-spanning membrane proteins are commonly viewed as membrane anchors for functional domains. Influenza virus neuraminidase (NA) exemplifies this concept, as it retains enzymatic function upon proteolytic release from the membrane. However, the subtype 1 NA TMDs have become increasingly more polar in human strains since 1918, which suggests that selection pressure exists on this domain. Here, we investigated the N1 TMD-head domain relationship by exchanging a prototypical "old" TMD (1933) with a "recent" (2009), more polar TMD and an engineered hydrophobic TMD. Each exchange altered the TMD association, decreased the NA folding efficiency, and significantly reduced viral budding and replication at 37°C compared to at 33°C, at which NA folds more efficiently. Passaging the chimera viruses at 37°C restored the NA folding efficiency, viral budding, and infectivity by selecting for NA TMD mutations that correspond with their polar or hydrophobic assembly properties. These results demonstrate that single-spanning membrane protein TMDs can influence distal domain folding, as well as membrane-related processes, and suggest the NA TMD in H1N1 viruses has become more polar to maintain compatibility with the evolving enzymatic head domain. IMPORTANCE The neuraminidase (NA) protein from influenza A viruses (IAVs) functions to promote viral release and is one of the major surface antigens. The receptor-destroying activity in NA resides in the distal head domain that is linked to the viral membrane by an N-terminal hydrophobic transmembrane domain (TMD). Over the last century, the subtype 1 NA TMDs (N1) in human H1N1 viruses have become increasingly more polar, and the head domains have changed to alter their antigenicity. Here, we provide the first evidence that an "old" N1 head domain from 1933 is incompatible with a "recent" (2009), more polar N1 TMD sequence and that, during viral replication, the head domain drives the selection of TMD mutations. These mutations modify the intrinsic TMD assembly to restore the head domain folding compatibility and the resultant budding deficiency. This likely explains why the N1 TMDs have become more polar and suggests the N1 TMD and head domain have coevolved. Receptors on the surface of cells and enveloped viruses are often comprised of single-spanning (bitopic) membrane proteins. The majority of the structures for these proteins exclude their transmembrane domain (TMD), which has hindered the ability to identify any potential contributions of the TMD to protein folding and activity. Instead, studies with model and natural TMD segments in reductionist systems have been used to determine how they interact (reviewed in references 1-3). The biological functions of these TMD interactions in bitopic proteins are much less characterized and have been linked mainly to oligomerization and activity (4-9). The current challenge is to better define the plasticity of bitopic TMD interactions and how they contribute to protein folding and activity in n...

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Influenza A Virus Neuraminidase Inhibitors

Sriwilaijaroen

Vavricka

Kiyota

et al. 2022

Methods in Molecular Biology

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Assembly of Subtype 1 Influenza Neuraminidase Is Driven by Both the Transmembrane and Head Domains

Cited by 45 publications

References 49 publications

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

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

The Influenza Virus Neuraminidase Protein Transmembrane and Head Domains Have Coevolved

Influenza A Virus Neuraminidase Inhibitors

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