Tweet
J Virol. 2016 Aug 10. pii: JVI.01346-16. [Epub ahead of print]
Identification of residues that affect oligomerization and/or enzymatic activity of influenza virus H5N1 neuraminidase proteins.
Dai M1, Guo H1, Dortmans J1, Dekkers J1, Nordholm J2, Daniels R2, van Kuppeveld FJ1, de Vries E1, de Haan CA3.
Author information
Abstract
Influenza A virus attachment to and release from sialoside receptors is determined by the balance between hemagglutinin 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.
IMPORTANCE:
The IAV hemagglutinin (HA) and neuraminidase (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 that of full-length proteins compared to the other tetramerization domains used.
Copyright ? 2016, American Society for Microbiology. All Rights Reserved.
PMID: 27512075 DOI: 10.1128/JVI.01346-16
[PubMed - as supplied by publisher]
Identification of residues that affect oligomerization and/or enzymatic activity of influenza virus H5N1 neuraminidase proteins.
Dai M1, Guo H1, Dortmans J1, Dekkers J1, Nordholm J2, Daniels R2, van Kuppeveld FJ1, de Vries E1, de Haan CA3.
Author information
Abstract
Influenza A virus attachment to and release from sialoside receptors is determined by the balance between hemagglutinin 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.
IMPORTANCE:
The IAV hemagglutinin (HA) and neuraminidase (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 that of full-length proteins compared to the other tetramerization domains used.
Copyright ? 2016, American Society for Microbiology. All Rights Reserved.
PMID: 27512075 DOI: 10.1128/JVI.01346-16
[PubMed - as supplied by publisher]