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

Abstract

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...

Substrate Binding by the Second Sialic Acid-Binding Site of Influenza A Virus N1 Neuraminidase Contributes to Enzymatic Activity

“…Analysis of the relative specific activities of the different proteins when the MUNANA substrate was used indicated that the HN, TK, and HB proteins cleave this substrate 2to 3-fold more efficiently than the VN protein ( Fig. 1A), in agreement with previous results (22). A remarkably different result was obtained when the relative specific activity was determined with fetuin using ECA staining ( Fig.…”

“…Previously, we expressed recombinant soluble tetrameric versions of the N1 proteins derived from different highly pathogenic H5N1 viruses (A/duck/Hunan/795/2002, A/Vietnam/1194/04, A/turkey/Turkey/1/2005, and A/Hubei/1/2010, referred to as HN, VN, TK and HB viruses, respectively) and analyzed their enzymatic activities using the monovalent substrate MUNANA [2=-(4-methylumbelliferyl)-␣-D-N-acetylneuraminic acid] (22). In the current study, we analyzed the enzymatic activity of these proteins by enzyme-linked lectin assays (ELLAs) (10) using the glycoproteins fetuin and transferrin.…”

Mutation of the Second Sialic Acid-Binding Site, Resulting in Reduced Neuraminidase Activity, Preceded the Emergence of H7N9 Influenza A Virus

“…Previously, we compared the HA receptor-binding properties of a novel H7N9 virus isolated from a human patient in 2013 (A/Anhui/1/2013, referred to as "Anhui") with those of a closely related avian H7N9 virus from 2008 (A/Anas crecca/Spain/1460/ 2008, referred to as "Spain") (15). However, the NA stalk domain may contribute to correct folding (30) and affect enzymatic activity (31,32). Detailed analysis of the N9 proteins was precluded, however, by the low expression levels and activities of the recombinant soluble N9 proteins.…”

“…1A). We previously showed that replacing GCN4-pLI with a tetrabrachion tetramerization domain also resulted in NA proteins with higher specific activity (31), and therefore, we made N9 constructs with such a tetrabrachion domain. Therefore, we constructed novel expression vectors encoding N9 protein head domains extended with their stalk sequences ( Fig.…”

“…Expression constructs of the recombinant soluble H7 and N9 proteins of A/Anhui/1/2013 (GISAID isolate EPI439507, referred to as H7 Anhui and N9 Anhui) and of A/Anas crecca/Spain/1460/2008 (GenBank accession no. Finally, the GCN4-pLI tetramerization domain was exchanged with the Staphylothermus marinus tetrabrachion tetramerization domain (31,48) (referred to as TE-NA). In these N9 expression constructs, sequences encoding the NA head domain (amino acids 76 to 470) of N9 Anhui and N9 Spain were preceded by sequences encoding a CD5 N-terminal signal sequence, a double Strep tag for purification (One-STrEP; IBA GmbH), and a GCN4-pLI (47) tetramerization domain (referred to as GCN4-NA head ).…”