scholarly journals Amino Acid Residues Contributing to the Substrate Specificity of the Influenza A Virus Neuraminidase

1999 ◽  
Vol 73 (8) ◽  
pp. 6743-6751 ◽  
Author(s):  
Darwyn Kobasa ◽  
Shantha Kodihalli ◽  
Ming Luo ◽  
Maria R. Castrucci ◽  
Isabella Donatelli ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Viral Replication ◽  
Active Site ◽  
Influenza A ◽  
Sialic Acids ◽  
Influenza A Viruses ◽  
Human Viruses

ABSTRACT 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 betweenN-acetyl sialic acid and galactose (NeuAcα2-3Gal and NeuAcα2-6Gal) 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 NeuAcα2-3Gal, while the activities for NeuAcα2-6Gal ranged from marginal, as represented by avian and early N2 human viruses, to high (although only one-third the activity for NeuAcα2-3Gal), 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 NeuAcα2-6Gal 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.

scholarly journals Sialic Acid Species as a Determinant of the Host Range of Influenza A Viruses

2000 ◽  
Vol 74 (24) ◽  
pp. 11825-11831 ◽  
Author(s):  
Yasuo Suzuki ◽  
Toshihiro Ito ◽  
Takashi Suzuki ◽  
Robert E. Holland ◽  
Thomas M. Chambers ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Viral Replication ◽  
Influenza A ◽  
Lectin Binding ◽  
Influenza Viruses ◽  
Influenza A Viruses ◽  
Acetylneuraminic Acid ◽  
Human Viruses ◽  
Immunohistochemical Analyses

ABSTRACT The distribution of sialic acid (SA) species varies among animal species, but the biological role of this variation is largely unknown. Influenza viruses differ in their ability to recognize SA-galactose (Gal) linkages, depending on the animal hosts from which they are isolated. For example, human viruses preferentially recognize SA linked to Gal by the α2,6(SAα2,6Gal) linkage, while equine viruses favor SAα2,3Gal. However, whether a difference in relative abundance of specific SA species (N-acetylneuraminic acid [NeuAc] andN-glycolylneuraminic acid [NeuGc]) among different animals affects the replicative potential of influenza viruses is uncertain. We therefore examined the requirement for the hemagglutinin (HA) for support of viral replication in horses, using viruses whose HAs differ in receptor specificity. A virus with an HA recognizing NeuAcα2,6Gal but not NeuAcα2,3Gal or NeuGcα2,3Gal failed to replicate in horses, while one with an HA recognizing the NeuGcα2,3Gal moiety replicated in horses. Furthermore, biochemical and immunohistochemical analyses and a lectin-binding assay demonstrated the abundance of the NeuGcα2,3Gal moiety in epithelial cells of horse trachea, indicating that recognition of this moiety is critical for viral replication in horses. Thus, these results provide evidence of a biological effect of different SA species in different animals.

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

2018 ◽  
Vol 92 (20) ◽  
Author(s):  
Wenjuan Du ◽  
Meiling Dai ◽  
Zeshi Li ◽  
Geert-Jan Boons ◽  
Ben Peeters ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Enzymatic Activity ◽  
Binding Site ◽  
Influenza A Virus ◽  
Influenza A ◽  
Substrate Binding ◽  
Host Tropism ◽  
Contact Residues ◽  
Human Viruses

ABSTRACTThe influenza A virus (IAV) neuraminidase (NA) protein plays an essential role in the release of virus particles from cells and decoy receptors. The NA enzymatic activity presumably needs to match the activity of the IAV hemagglutinin (HA) attachment protein and the host sialic acid (SIA) receptor repertoire. We analyzed the enzymatic activities of N1 NA proteins derived from avian (H5N1) and human (H1N1) IAVs and analyzed the role of the second SIA-binding site, located adjacent to the conserved catalytic site, therein. SIA contact residues in the second SIA-binding site of NA are highly conserved in avian, but not human, IAVs. All N1 proteins preferred cleaving α2,3- over α2,6-linked SIAs even when their corresponding HA proteins displayed a strict preference for α2,6-linked SIAs, indicating that the specificity of the NA protein does not need to fully match that of the corresponding HA protein. NA activity was affected by substitutions in the second SIA-binding site that are observed in avian and human IAVs, at least when multivalent rather than monovalent substrates were used. These mutations included both SIA contact residues and residues that do not directly interact with SIA in all three loops of the second SIA-binding site. Substrate binding via the second SIA-binding site enhanced the catalytic activity of N1. Mutation of the second SIA-binding site was also shown to affect virus replicationin vitro. Our results indicate an important role for the N1 second SIA-binding site in binding to and cleavage of multivalent substrates.IMPORTANCEAvian and human influenza A viruses (IAVs) preferentially bind α2,3- and α2,6-linked sialic acids (SIAs), respectively. A functional balance between the hemagglutinin (HA) attachment and neuraminidase (NA) proteins is thought to be important for host tropism. What this balance entails at the molecular level is, however, not well understood. We now show that N1 proteins of both avian and human viruses prefer cleaving avian- over human-type receptors although human viruses were relatively better in cleavage of the human-type receptors. In addition, we show that substitutions at different positions in the second SIA-binding site found in NA proteins of human IAVs have a profound effect on binding and cleavage of multivalent, but not monovalent, receptors and affect virus replication. Our results indicate that the HA-NA balance can be tuned via modification of substrate binding via this site and suggest an important role of the second SIA-binding site in host tropism.

Investigating the role of active site residues of Rhodotorula gracilis d-amino acid oxidase on its substrate specificity

Biochimie ◽  
2007 ◽  
Vol 89 (3) ◽  
pp. 360-368 ◽  
Author(s):  
Angelo Boselli ◽  
Luciano Piubelli ◽  
Gianluca Molla ◽  
Mirella S. Pilone ◽  
Loredano Pollegioni ◽  
...  
Keyword(s):  
Amino Acid ◽  
Substrate Specificity ◽  
Active Site ◽  
Acid Oxidase ◽  
Active Site Residues ◽  
Amino Acid Oxidase ◽  
Rhodotorula Gracilis

scholarly journals The Low-pH Resistance of Neuraminidase Is Essential for the Replication of Influenza A Virus in Duck Intestine following Infection via the Oral Route

2016 ◽  
Vol 90 (8) ◽  
pp. 4127-4132 ◽  
Author(s):  
Yoshikazu Fujimoto ◽  
Hiroshi Ito ◽  
Etsuro Ono ◽  
Yoshihiro Kawaoka ◽  
Toshihiro Ito
Keyword(s):  
Amino Acid ◽  
Viral Replication ◽  
Influenza A Virus ◽  
Influenza A ◽  
Reverse Genetics ◽  
Biological Properties ◽  
Oral Route ◽  
Low Ph ◽  
Amino Acid Residues ◽  
Influenza A Viruses

ABSTRACTInfluenza A viruses are known to primarily replicate in duck intestine following infection via the oral route, but the specific role of neuraminidase (NA) for the intestinal tropism of influenza A viruses has been unclear. A reassortant virus (Dk78/Eng62N2) did not propagate in ducks infected via the oral route. To generate variant viruses that grow well in ducks via the oral route, we isolated viruses that effectively replicate in intestinal mucosal cells by passaging Dk78/Eng62N2 in duck via rectal-route infection. This procedure led to the isolation of a variant virus from the duck intestine. This virus was propagated using embryonated chicken eggs and inoculated into a duck via the oral route, which led to the isolation of Dk-rec6 from the duck intestine. Experimental infections with mutant viruses generated by using reverse genetics indicated that the paired mutation of residues 356 and 431 in NA was necessary for the viral replication in duck intestine. The NA assay revealed that the activity of Dk78/Eng62N2 almost disappeared after pH 3 treatment, whereas that of Dk-rec6 was maintained. Furthermore, to identify the amino acid residues associated with the low-pH resistance, we measured the activities of mutant NA proteins transiently expressed in 293 cells after pH 3 treatment. All mutant NA proteins that possessed proline at position 431 showed higher activities than NA proteins that possessed glutamine at this position. These findings indicate that the low-pH resistance of NA plays an important role in the ability of influenza A virus to replicate in duck intestine.IMPORTANCENeuraminidase (NA) activity facilitates the release of viruses from cells and, as such, is important for the replicative efficiency of influenza A virus. Ducks are believed to serve as the principal natural reservoir for influenza A virus; however, the key properties of NA for viral infection in duck are not well understood. In this study, we identify amino acid residues in NA that contribute to viral replication in ducks via the natural route of infection and demonstrate that maintenance of NA activity under low-pH conditions is associated with the biological properties of the virus. These findings provide insights into the mechanisms of replication of influenza A virus in ducks.

scholarly journals S-Acylation of Proteins of Coronavirus and Influenza Virus: Conservation of Acylation Sites in Animal Viruses and DHHC Acyltransferases in Their Animal Reservoirs

Pathogens ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 669
Author(s):  
Dina A. Abdulrahman ◽  
Xiaorong Meng ◽  
Michael Veit
Keyword(s):  
Influenza Virus ◽  
Amino Acid ◽  
Ion Channels ◽  
Substrate Specificity ◽  
Influenza A ◽  
3D Structure ◽  
Viral Fitness ◽  
Amino Acid Substitutions ◽  
Essential Function ◽  
Animal Reservoirs

Recent pandemics of zoonotic origin were caused by members of coronavirus (CoV) and influenza A (Flu A) viruses. Their glycoproteins (S in CoV, HA in Flu A) and ion channels (E in CoV, M2 in Flu A) are S-acylated. We show that viruses of all genera and from all hosts contain clusters of acylated cysteines in HA, S and E, consistent with the essential function of the modification. In contrast, some Flu viruses lost the acylated cysteine in M2 during evolution, suggesting that it does not affect viral fitness. Members of the DHHC family catalyze palmitoylation. Twenty-three DHHCs exist in humans, but the number varies between vertebrates. SARS-CoV-2 and Flu A proteins are acylated by an overlapping set of DHHCs in human cells. We show that these DHHC genes also exist in other virus hosts. Localization of amino acid substitutions in the 3D structure of DHHCs provided no evidence that their activity or substrate specificity is disturbed. We speculate that newly emerged CoVs or Flu viruses also depend on S-acylation for replication and will use the human DHHCs for that purpose. This feature makes these DHHCs attractive targets for pan-antiviral drugs.

scholarly journals Characterization of H9N2 influenza A viruses isolated from chicken products imported into Japan from China

2006 ◽  
Vol 135 (3) ◽  
pp. 386-391 ◽  
Author(s):  
M. MASE ◽  
M. ETO ◽  
K. IMAI ◽  
K. TSUKAMOTO ◽  
S. YAMAGUCHI
Keyword(s):  
Influenza Virus ◽  
Amino Acid ◽  
Avian Influenza ◽  
Avian Influenza Virus ◽  
Influenza A ◽  
H9n2 Virus ◽  
Amino Acid Substitutions ◽  
Influenza A Viruses ◽  
Potential Sources

We characterized eleven H9N2 influenza A viruses isolated from chicken products imported from China. Genetically they were classified into six distinct genotypes, including five already known genotypes and one novel genotype. This suggested that such multiple genotypes of the H9N2 virus have possibly already become widespread and endemic in China. Two isolates have amino-acid substitutions that confer resistance to amantadine in the M2 region, and this supported the evidence that this mutation might be a result of the wide application of amantadine for avian influenza treatment in China. These findings emphasize the importance of surveillance for avian influenza virus in this region, and of quarantining imported chicken products as potential sources for the introduction of influenza virus.

scholarly journals Functional Analysis of PA Binding by Influenza A Virus PB1: Effects on Polymerase Activity and Viral Infectivity

2001 ◽  
Vol 75 (17) ◽  
pp. 8127-8136 ◽  
Author(s):  
Daniel R. Perez ◽  
Ruben O. Donis
Keyword(s):  
Amino Acids ◽  
Influenza Virus ◽  
Amino Acid ◽  
Viral Replication ◽  
Influenza A Virus ◽  
Influenza A ◽  
Amino Acid Sequences ◽  
Influenza Viruses ◽  
Virus Growth ◽  
Transcription And Replication

ABSTRACT Influenza A virus expresses three viral polymerase (P) subunits—PB1, PB2, and PA—all of which are essential for RNA and viral replication. The functions of P proteins in transcription and replication have been partially elucidated, yet some of these functions seem to be dependent on the formation of a heterotrimer for optimal viral RNA transcription and replication. Although it is conceivable that heterotrimer subunit interactions may allow a more efficient catalysis, direct evidence of their essentiality for viral replication is lacking. Biochemical studies addressing the molecular anatomy of the P complexes have revealed direct interactions between PB1 and PB2 as well as between PB1 and PA. Previous studies have shown that the N-terminal 48 amino acids of PB1, termed domain α, contain the residues required for binding PA. We report here the refined mapping of the amino acid sequences within this small region of PB1 that are indispensable for binding PA by deletion mutagenesis of PB1 in a two-hybrid assay. Subsequently, we used site-directed mutagenesis to identify the critical amino acid residues of PB1 for interaction with PA in vivo. The first 12 amino acids of PB1 were found to constitute the core of the interaction interface, thus narrowing the previous boundaries of domain α. The role of the minimal PB1 domain α in influenza virus gene expression and genome replication was subsequently analyzed by evaluating the activity of a set of PB1 mutants in a model reporter minigenome system. A strong correlation was observed between a functional PA binding site on PB1 and P activity. Influenza viruses bearing mutant PB1 genes were recovered using a plasmid-based influenza virus reverse genetics system. Interestingly, mutations that rendered PB1 unable to bind PA were either nonviable or severely growth impaired. These data are consistent with an essential role for the N terminus of PB1 in binding PA, P activity, and virus growth.

scholarly journals N-Glycolylneuraminic Acid in Animal Models for Human Influenza A Virus

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 815
Author(s):  
Cindy M. Spruit ◽  
Nikoloz Nemanichvili ◽  
Masatoshi Okamatsu ◽  
Hiromu Takematsu ◽  
Geert-Jan Boons ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Animal Models ◽  
Influenza A ◽  
Influenza Virus Infection ◽  
Influenza Viruses ◽  
Upper Respiratory Tract ◽  
Human Influenza ◽  
Influenza A Viruses ◽  
Sialic Acid Content ◽  
Acetylneuraminic Acid

The first step in influenza virus infection is the binding of hemagglutinin to sialic acid-containing glycans present on the cell surface. Over 50 different sialic acid modifications are known, of which N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) are the two main species. Animal models with α2,6 linked Neu5Ac in the upper respiratory tract, similar to humans, are preferred to enable and mimic infection with unadapted human influenza A viruses. Animal models that are currently most often used to study human influenza are mice and ferrets. Additionally, guinea pigs, cotton rats, Syrian hamsters, tree shrews, domestic swine, and non-human primates (macaques and marmosets) are discussed. The presence of NeuGc and the distribution of sialic acid linkages in the most commonly used models is summarized and experimentally determined. We also evaluated the role of Neu5Gc in infection using Neu5Gc binding viruses and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH)-/- knockout mice, which lack Neu5Gc and concluded that Neu5Gc is unlikely to be a decoy receptor. This article provides a base for choosing an appropriate animal model. Although mice are one of the most favored models, they are hardly naturally susceptible to infection with human influenza viruses, possibly because they express mainly α2,3 linked sialic acids with both Neu5Ac and Neu5Gc modifications. We suggest using ferrets, which resemble humans closely in the sialic acid content, both in the linkages and the lack of Neu5Gc, lung organization, susceptibility, and disease pathogenesis.

scholarly journals Plasticity of the Influenza Virus H5 HA Protein

mBio ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Huihui Kong ◽  
David F. Burke ◽  
Tiago Jose da Silva Lopes ◽  
Kosuke Takada ◽  
Masaki Imai ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Amino Acid ◽  
Mammalian Cells ◽  
Influenza A ◽  
Viral Antigen ◽  
Influenza Viruses ◽  
Influenza A Viruses ◽  
Highly Pathogenic ◽  
Antigenic Properties ◽  
Ha Protein

ABSTRACT Since the emergence of highly pathogenic avian influenza viruses of the H5 subtype, the major viral antigen, hemagglutinin (HA), has undergone constant evolution, resulting in numerous genetic and antigenic (sub)clades. To explore the consequences of amino acid changes at sites that may affect the antigenicity of H5 viruses, we simultaneously mutated 17 amino acid positions of an H5 HA by using a synthetic gene library that, theoretically, encodes all combinations of the 20 amino acids at the 17 positions. All 251 mutant viruses sequenced possessed ≥13 amino acid substitutions in HA, demonstrating that the targeted sites can accommodate a substantial number of mutations. Selection with ferret sera raised against H5 viruses of different clades resulted in the isolation of 39 genotypes. Further analysis of seven variants demonstrated that they were antigenically different from the parental virus and replicated efficiently in mammalian cells. Our data demonstrate the substantial plasticity of the influenza virus H5 HA protein, which may lead to novel antigenic variants. IMPORTANCE The HA protein of influenza A viruses is the major viral antigen. In this study, we simultaneously introduced mutations at 17 amino acid positions of an H5 HA expected to affect antigenicity. Viruses with ≥13 amino acid changes in HA were viable, and some had altered antigenic properties. H5 HA can therefore accommodate many mutations in regions that affect antigenicity. The substantial plasticity of H5 HA may facilitate the emergence of novel antigenic variants.

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