Virus Sialidase Research Articles

Synthetic Sialosides Terminated with 8‐N‐Substituted Sialic Acid as Selective Substrates for Sialidases from Bacteria and Influenza Viruses

AbstractSialosides containing C8‐modified sialic acids are challenging synthetic targets but potentially useful probes for diagnostic substrate profiling of sialidases and elucidating the binding specificity of sialic acid‐interacting proteins. Here, we demonstrate efficient chemoenzymatic methods for synthesizing para‐nitrophenol‐tagged α2–3‐ and α2–6‐linked sialyl galactosides containing C8‐acetamido, C8‐azido, or C8‐amino derivatized N‐acetylneuraminic acid (Neu5Ac). High‐throughput substrate specificity studies showed that the C8‐modification of sialic acid significantly changes its recognition by sialidases from humans, various bacteria, and different influenza A and B viruses. Sialosides carrying Neu5Ac with a C8‐azido modification were generally well tolerated by all the sialidases we tested, whereas sialosides containing C8‐acetamido‐modified Neu5Ac were only cleaved by selective bacterial sialidases. In contrast, sialosides with C8‐amino‐modified Neu5Ac were cleaved by a combination of selective bacterial and influenza A virus sialidases. These results indicate that sialosides terminated with a C8‐amino or C8‐acetamido‐modified sialic acid can be used with other sialosides for diagnostic profiling of disease‐causing sialidase‐producing pathogens.

Synthetic Sialosides Terminated with 8-N-Substituted Sialic Acid as Selective Substrates for Sialidases from Bacteria and Influenza Viruses.

Sialosides containing C8-modified sialic acids are challenging synthetic targets but potentially useful probes for diagnostic substrate profiling of sialidases and elucidating the binding specificity of sialic acid-interacting proteins. Here, we demonstrate efficient chemoenzymatic methods for synthesizing para-nitrophenol-tagged α2-3- and α2-6-linked sialyl galactosides containing C8-acetamido, C8-azido, or C8-amino derivatized N-acetylneuraminic acid (Neu5Ac). High-throughput substrate specificity studies showed that the C8-modification of sialic acid significantly changes its recognition by sialidases from humans, various bacteria, and different influenza A and B viruses. Sialosides carrying Neu5Ac with a C8-azido modification were generally well tolerated by all the sialidases we tested, whereas sialosides containing C8-acetamido-modified Neu5Ac were only cleaved by selective bacterial sialidases. In contrast, sialosides with C8-amino-modified Neu5Ac were cleaved by a combination of selective bacterial and influenza A virus sialidases. These results indicate that sialosides terminated with a C8-amino or C8-acetamido-modified sialic acid can be used with other sialosides for diagnostic profiling of disease-causing sialidase-producing pathogens.

Effective inhibition of bacterial sialidases by phenolic acids and flavonoids

As a pathogenicity factor in some microorganisms, sialidase is a key target for inactivation, as this would have curative and preventive effects on various diseases. Significant results are already achieved with viral sialidase inhibitors, while such studies on bacterial enzymes are scarce. Pure natural compounds representing phenols and flavonoids, were tested for their inhibitory effect on sialidases from Vibrio cholerae non-O1, Arthrobacter nicotianae and Oerskovia paurometabola. All three enzymes were isolated, purified beforehand and stored under suitable conditions. Quinic and gallic acids showed the highest inhibitory activity - 76 to 100% against the three sialidases. Fisetin had a significant inhibitory activity on two of the enzymes. The structurally related thymol and thymoquinone exerted from 80 to 100% inhibition on at least one of the enzymes. Catechin and rutin had significant inhibitory activity, varying from 49 to 100%, on some of the enzymes. Quercetin, known for its inhibitory effect on viral sialidases, had a lesser impact on the studied enzymes. The suppressive effect of quinic acid, rutin and fisetin on bacterial sialidases is observed for the first time.

Visualizing intracellular sialidase activity of influenza A virus neuraminidase using a fluorescence imaging probe

In influenza A virus-infected cells, newly synthesized viral neuraminidases (NAs) transiently localize at the host cell Golgi due to glycosylation, before their expression on the cell surface. It remains unproven whether Golgi-localized intracellular NAs exhibit sialidase activity. We have developed a sialidase imaging probe, [2-(benzothiazol-2-yl)−5-(non-1-yn-1-yl) phenyl]-α-D-N-acetylneuraminic acid (BTP9-Neu5Ac). This probe is designed to be cleaved by sialidase activity, resulting in the release of a hydrophobic fluorescent compound, 2-(benzothiazol-2-yl)−5-(non-1-yn-1-yl) phenol (BTP9). BTP9-Neu5Ac makes the location of sialidase activity visually detectable by the BTP9 fluorescence that results from the action of sialidase activity. In this study, we established a protocol to visualize the sialidase activity of intracellular NA at the Golgi of influenza A virus-infected cells using BTP9-Neu5Ac. Furthermore, we employed this fluorescence imaging protocol to elucidate the intracellular inhibition of laninamivir octanoate, an anti-influenza drug. At approximately 7 h after infection, newly synthesized viral NAs localized at the Golgi. Using our developed protocol, we successfully histochemically stained the sialidase activity of intracellular viral NAs localized at the Golgi. Importantly, we observed that laninamivir octanoate effectively inhibited the intracellular viral NA, in contrast to drugs like zanamivir or laninamivir. Our study establishes a visualization protocol for intracellular viral NA sialidase activity and visualizes the inhibitory effect of laninamivir octanoate on Golgi-localized intracellular viral NA in infected cells.

Sialidase Inhibitors with Different Mechanisms.

Sialidases, or neuraminidases, are enzymes that catalyze the hydrolysis of sialic acid (Sia)-containing molecules, mostly removal of the terminal Sia (desialylation). By desialylation, sialidase can modulate the functionality of the target compound and is thus often involved in biological pathways. Inhibition of sialidases with inhibitors is an important approach for understanding sialidase function and the underlying mechanisms and could serve as a therapeutic approach as well. Transition-state analogues, such as anti-influenza drugs oseltamivir and zanamivir, are major sialidase inhibitors. In addition, difluoro-sialic acids were developed as mechanism-based sialidase inhibitors. Further, fluorinated quinone methide-based suicide substrates were reported. Sialidase product analogue inhibitors were also explored. Finally, natural products have shown competitive inhibiton against viral, bacterial, and human sialidases. This Perspective describes sialidase inhibitors with different mechanisms and their activities and future potential, which include transition-state analogue inhibitors, mechanism-based inhibitors, suicide substrate inhibitors, product analogue inhibitors, and natural product inhibitors.

Analysis and Control of Viral Infection Mechanisms by Glycobiology

Glycans are present in all living organisms, including viruses, bacteria, and animals, and perform various biological functions. The author has been studying influenza viruses' glycan usage mechanisms, particularly the functional analysis of neuraminidase (NA), a viral sialidase. The authors recently focused on influenza virus NAs' high sialidase activity with the aim of using sialidase activity detection as a virus detection technology. Using the probe BTP3-Neu5Ac, allows fluorescent imaging of sialidase activity, we created a new technique for easy, rapid, and high sensitivity fluorescent imaging of virus-infected cells. The detection of viruses using BTP3-Neu5Ac does not require specific antibodies and can be performed by simply adding reagents. Furthermore, fluorescence imaging of sialidase as a virus detection technology has many advantages, including isolating viruses from fluorescently imaged infected cells. This detection technique is easy to use in basic research and hygiene testing, where viral culture is conducted and is expected to be widely used.

The genome and phylogenetic analyses of tit siadenoviruses reveal both a novel avian host and viral species.

In both a Eurasian blue tit (Cyanistes caeruleus) and a great tit (Parus major), found dead in North Rhine-Westphalia, Germany, intranuclear inclusion bodies were observed in the kidneys during the histologic examination. Siadenoviruses were detected in both samples, and the nucleotide sequence of the partial DNA polymerase, obtained from the blue tit, was almost identical with that of great tit adenovirus type 1, reported from Hungary previously. The sequence, derived from the German great tit sample was more similar to great tit adenovirus 2, yet divergent enough to forecast the possible establishment of a novel viral type and species. Therefore, the complete genome was subjected to next generation sequencing. The annotation revealed a typical siadenoviral genome layout, and phylogenetic analyses proved the distinctness of the novel virus type: great tit adenovirus 3. We propose the establishment of a new species (Siadenovirus carbocapituli) within the genus Siadenovirus to contain great tit adenovirus types 2 and 3. As both of the newly-detected viruses originated from histologically confirmed cases, and several siadenoviruses have been associated with avian nephritis earlier, we assume that the renal pathology might have been also of adenoviral origin. Additionally, we performed structural studies on two virus-coded proteins. The viral sialidase and the fiber knob were modeled using the AlphaFold2 program. According to the results of the sialic acid docking studies, the fiber trimer of the new great tit adenovirus 3 binds this acid with good affinity. As sialic acid is expressed in the kidney, it can be hypothesized that it is used during the receptor binding and entry of the virus. Strong binding of sialic acid was also predictable for the viral sialidase albeit its enzymatic activity remains disputable since, within its catalytic site, an asparagine residue was revealed instead of the conserved aspartic acid.

Viral sialidases and atherosclerosis

Background and Aims : Sialidases play an important role in atherosclerosis development due to modification of low-density lipoproteins (LDL). Removal of sialic acid residues from native LDL leads to the formation of atherogenic desialylated LDL. But the exact details of the formation of these modified LDL are still to be clarified. The aim of this study was to explore the contribution of viral sialidases to the total sialidase activity in blood plasma.

The antiviral effect of catechins on mumps virus infection

• The inhibitory effect and its mechanism of catechins on mumps virus infection. • Epigallocatechin gallate shows the highest antiviral potency. • A galloyl group in catechins is an important factor for the strong antiviral effect. • The antiviral effect can result from inhibition of the viral fusion activity. Catechins have been known to possess antiviral effects against influenza A and B viruses through the inhibition of both the viral receptor binding and viral sialidase activities. Here, we examined the antiviral effect of catechins on mumps virus infection. The addition of epigallocatechin gallate (EGCG), gallocatechin gallate, epicatechin gallate, or catechin gallate simultaneously with the mumps virus infection of Vero cells greatly inhibited the infection. EGCG showed the strongest antiviral effect among the catechins tested. These catechins did not exhibit an inhibitory effect against the receptor binding and sialidase activities in the hemagglutinin–neuraminidase of the mumps virus, which was different from a known mechanism for influenza A and B viruses. By contrast, these catechins inhibited the viral fusion activity. Our results indicate that catechins are a useful food-derived antiviral agent against the mumps virus.

Study of the effect of the sialidase inhibitor oseltamivir phosphate on sialidase activity in the blood.

Background and Aims: The aim of the study was to establish the possible role of viral sialidases in desialation of low-density LDL and the way of therapeutic correction of these changes.

Sialidase Activity in Human Blood Serum Has a Distinct Seasonal Pattern: A Pilot Study.

Desialylation—loss of terminal sialic acid residues from glycoconjugates catalyzed by sialidases—is involved in many human diseases and is considered a key molecular event of atherosclerosis onset. Desialylated low-density lipoproteins with atherogenic properties have been detected in human blood previously. However, there is currently no consensus on the origin of desialylation activity in the bloodstream. Here, we suggest viral intervention as a possible explanation. In order to address our hypothesis, we studied seasonal patterns of blood serum sialidase enzymatic activity and designed an approach to detect and quantify viral sialidase genetic presence. Increased sialidase activity in autumn-winter combined with detectable levels of influenza virus sialidase mRNA suggests exogenous viral sialidase as a viable component of desialylation in human blood, providing new insights on the molecular background of atherogenesis.

Investigation of substrate specificity of sialidases with membrane mimetic glycoconjugates.

Sialidases or neuraminidases play important roles in various physiological and pathological processes by cleaving terminal sialic acids (Sias) (desialylation) from the glycans of both glycoproteins and glycolipids. To understand the biological significance of desialylation by sialidases, it is important to investigate enzyme specificity with native substrate in biological membrane of cells. Herein, we report a membrane-mimicking system with liposome ganglioside conjugates containing different lipids for evaluating substrate specificity of sialidase and the lipid effect on the enzyme activity. Briefly, liposomes of phosphatidylcholine (PC) and cholesterol with ganglioside (GM3 or GM1) along with different percentage of phosphatidylserine (PS) or phosphatidylethanolamine (PE) were prepared and characterized. Their desialylation profiles with Arthrobacter ureafaciens (bacterial) sialidase and H1N1 (influenza viral) sialidase were quantified by HPLC method. A diversity of substrate preference was found for both bacterial and viral sialidase to the liposome ganglioside conjugate platform. The apparent Km and Vmax were dependent on the type of lipid. These results indicate that the intrinsic characteristics of the membrane-like system affect the sialidase specificity and activity. This biomimetic substrate provides a better tool for unravelling the substrate specificity and the biological function of sialidases and for screening of functional sialidase inhibitors as well.

Fluorogenic Probes for Accurate in Situ Imaging of Viral and Mammalian Sialidases.

Sialidases are widely distributed in nature and are involved in many physiological and pathological processes. Sialidases are expressed and work in various tissues and organelles. Clarification of the localization of sialidases is very helpful as a way to understand their functions. We previously developed a novel fluorogenic probe for sialidases, BTP3-Neu5Ac, that visualized the localization of sialidase activity in live cells and tissues by precipitating the hydrophobic fluorescent compound; however, for the purpose of accurate fluorescence imaging of sialidase-expressing cells or the distribution of intracellular sialidase activity, BTP3-Neu5Ac was inadequate in imaging performance. We report the design and development of a sialidase imaging probe that improves the sensitivity and accuracy of in situ fluorescence imaging performance as well as increases the hydrophobicity by attaching linear unsaturated hydrocarbon chains into the hydrophobic fluorescent compound of BTP3-Neu5Ac. The newly developed probe showed low diffusivity and high brightness for fluorescence imaging, and it enabled sensitive and highly accurate imaging of viral sialidase in virus-infected cells and sialidase-expressing cells as well as mammalian sialidase in the rat brain. The probe also enabled the fluorescence imaging of intracellular viral sialidase in live-virus-infected cells. The newly developed probe is expected to be a useful tool that will contribute to the progress of research on sialidases in various fields such as research on viruses and brains.

9-Azido-9-deoxy-2,3-difluorosialic Acid as a Subnanomolar Inhibitor against Bacterial Sialidases.

A library of 2(a),3(a/e)-difluorosialic acids and their C-5 and/or C-9 derivatives were chemoenzymatically synthesized. Pasteurella multocida sialic acid aldolase (PmAldolase), but not its Escherichia coli homologue (EcAldolase), was found to catalyze the formation of C5-azido analogue of 3-fluoro(a)-sialic acid. In comparison, both PmAldolase and EcAldolase could catalyze the synthesis of 3-fluoro(a/e)-sialic acids and their C-9 analogues although PmAldolase was generally more efficient. The chemoenzymatically synthesized 3-fluoro(a/e)-sialic acid analogues were purified and chemically derivatized to form the desired difluorosialic acids and derivatives. Inhibition studies against several bacterial sialidases and a recombinant human cytosolic sialidase hNEU2 indicated that sialidase inhibition was affected by the C-3 fluorine stereochemistry and derivatization at C-5 and/or C-9 of the inhibitor. Opposite to that observed for influenza A virus sialidases and hNEU2, compounds with axial fluorine at C-3 were better inhibitors (up to 100-fold) against bacterial sialidases compared to their 3F-equatorial counterparts. While C-5-modified compounds were less-efficient antibacterial sialidase inhibitors, 9-N3-modified 2,3-difluoro-Neu5Ac showed increased inhibitory activity against bacterial sialidases. 9-Azido-9-deoxy-2-(e)-3-(a)-difluoro- N-acetylneuraminic acid [2(e)3(a)DFNeu5Ac9N3] was identified as an effective inhibitor with a long effective duration selectively against pathogenic bacterial sialidases from Clostridium perfringens (CpNanI) and Vibrio cholerae.

Inhibition of sialidase activity as a therapeutic approach.

The demand for novel anti-influenza drugs persists, which is highlighted by the recent pandemics of influenza affecting thousands of people across the globe. One of the approaches to block the virus spreading is inhibiting viral sialidase (neuraminidase). This enzyme cleaves the sialic acid link between the newly formed virions and the host cell surface liberating the virions from the cell and maintaining the cycle of infection. Viral neuraminidases appear therefore as attractive therapeutic targets for preventing further spread of influenza infection. Compared to ion channel blockers that were the first approved anti-influenza drugs, neuraminidase inhibitors are well tolerated and target both influenza A and B viruses. Moreover, neuraminidase/sialidase inhibitors may be useful for managing some other human pathologies, such as cancer. In this review, we discuss the available knowledge on neuraminidase or sialidase inhibitors, their design, clinical application, and the current challenges.

Evaluation of the potential defensive strategy against Influenza A in cell line models.

Background: Influenza virus can cause both seasonal infections and unpredictable pandemics. Rapidly evolving avian H5N1 and H7N9 viruses have a potential pandemic threat for humans. Since avian Influenza can be transmitted by domestic birds, serving as a key link between wild birds and humans, an effective measure to control the influenza transmission would be eradication of the infection in poultry. It is known that the virus penetrates into the cell through binding with the terminal oligosaccharides - sialic acids (SA) - on the cell surfaces. Removal of SA might be a potential antiviral strategy. An approach to developing chicken lines that are resistant to influenza viruses could be the creation of genetically modified birds. Thus it is necessary to select a gene that provides defense to influenza. Here we have expressed in cells a range of exogenous sialidases and estimated their activity and specificity towards SA residues. Methods: Several bacterial, viral and human sialidases were tested. We adopted bacterial sialidases from Salmonella and Actinomyces for expression on the cell surface by fusing catalytic domains with transmembrane domains. We also selected Influenza A/PuertoRico/8/34/H1N1 neuraminidase and human membrane sialidase ( hNeu3) genes. Lectin binding assay was used for estimation of a α (2,3)-sialylation level by fluorescent microscopy and FACS. Results: We compared sialidases from bacteria, Influenza virus and human. Sialidases from Salmonella and Influenza A neuraminidase effectively cleaved α (2-3)-SA receptors. Viral neuraminidase demonstrated a higher activity. Sialidases from Actinomyces and hNeu3 did not show any activity against α (2-3) SA under physiological conditions. Conclusion: Our results demonstrated that sialidases with different specificity and activity can be selected as genes providing antiviral defence. Combining chosen sialidases with different activity together with tissue-specific promoters would provide an optimal level of desialylation. Tissue specific expression of the sialidases could protect domestic birds from infection.

Evaluation of the potential defensive strategy against Influenza A in cell line models

Background: Influenza virus can cause both seasonal infections and unpredictable pandemics. Rapidly evolving avian H5N1 and H7N9 viruses have a potential pandemic threat for humans. Since avian Influenza can be transmitted by domestic birds, serving as a key link between wild birds and humans, an effective measure to control the influenza transmission would be eradication of the infection in poultry. It is known that the virus penetrates into the cell through binding with the terminal oligosaccharides - sialic acids (SA) - on the cell surfaces. Removal of SA might be a potential antiviral strategy. An approach to developing chicken lines that are resistant to influenza viruses could be the creation of genetically modified birds. Thus it is necessary to select a gene that provides defense to influenza. Here we have expressed in cells a range of exogenous sialidases and estimated their activity and specificity towards SA residues. Methods: Several bacterial, viral and human sialidases were tested. We adopted bacterial sialidases from Salmonella and Actinomyces for expression on the cell surface by fusing catalytic domains with transmembrane domains. We also selected Influenza A/PuertoRico/8/34/H1N1 neuraminidase and human membrane sialidase ( hNeu3) genes. Lectin binding assay was used for estimation of a α (2,3)-sialylation level by fluorescent microscopy and FACS. Results: We compared sialidases from bacteria, Influenza virus and human. Sialidases from Salmonella and Influenza A neuraminidase effectively cleaved α (2-3)-SA receptors. Viral neuraminidase demonstrated a higher activity. Sialidases from Actinomyces and hNeu3 did not show any activity against α (2-3) SA under physiological conditions. Conclusion: Our results demonstrated that sialidases with different specificity and activity can be selected as genes providing antiviral defence. Combining chosen sialidases with different activity together with tissue-specific promoters would provide an optimal level of desialylation. Tissue specific expression of the sialidases could protect domestic birds from infection.

A Sulfonozanamivir Analogue Has Potent Anti-influenza Virus Activity.

Influenza virus infection continues to cause significant, often severe, respiratory illness worldwide. A validated target for the development of anti-influenza agents is the virus surface protein sialidase. In the current study, we have discovered a highly potent inhibitor of influenza virus sialidase, based on a novel sialosyl sulfonate template. The synthesised 3-guanidino sialosyl α-sulfonate, a sulfonozanamivir analogue, inhibits viral replication in vitro at the nanomolar level, comparable to that of the anti-influenza drug zanamivir. Using protein X-ray crystallography we show that the sialosyl α-sulfonate template binds within the sialidase active site in a 1 C4 chair conformation. The C1-sulfonate moiety forms crucial and strong-binding interactions with the active site's triarginyl cluster, while the 3-guanidino moiety interacts significantly with conserved active site residues. This sulfonozanamivir analogue provides a new direction in anti-influenza virus drug development.

Evaluation of defense strategy against Influenza A in cell line models

Background: Influenza virus can cause both seasonal infections and unpredictable pandemics. Rapidly evolving avian H5N1 virus is getting increasingly infective for humans. Since avian Influenza can be transmitted by domestic birds, serving as a key link between wild aquatic birds and humans, an effective measure to control the influenza transmission would be eradication of the infection in poultry. It is known that the virus penetrates into the cell through binding with the terminal oligosaccharides - sialic acids (SA) - on the cell surfaces. Removal of SA might be a potential antiviral strategy. An approach to developing chicken lines that are resistant to influenza viruses could be the creation of genetically modified birds. Thus it is necessary to select a gene that provides defense to influenza. Here we have expressed in cells a range of exogenous sialidases and estimated their activity and specificity towards SA residues. Methods: Several bacterial, viral and human sialidases were tested. We adopted bacterial sialidases from Salmonella and Actinomyces for expression on the cell surface by fusing catalytic domains with transmembrane domains. We also selected Influenza A/PuertoRico/8/34/H1N1 neuraminidase and human membrane sialidase ( hNeu3) genes. Lectin binding assay was used for estimation of a α (2,3)-sialylation level by fluorescent microscopy and FACS. Results: We compared sialidases from bacteria, Influenza virus and human. Sialidases from Salmonella and Influenza A neuraminidase effectively cleaved α (2-3)-SA receptors. Viral neuraminidase demonstrated a higher activity. Sialidases from Actinomyces and hNeu3 did not show any activity against α (2-3) SA under physiological conditions. Conclusion: Our results demonstrated that sialidases with different specificity and activity can be selected as genes providing antiviral defence. Combining chosen sialidases with different activity together with tissue-specific promoters would provide an optimal level of desialilation to prevent infection. Tissue specific expression of the sialidases could protect domestic birds from infection.

Synthesis and anti-influenza virus evaluation of triterpene-sialic acid conjugates

We are interested in new non-natural glycosides with sialic acid conjugates and their biological activities. We report the synthesis of eleven non-natural occurring glycosides, which are triterpene (glycyrrhetinic acid and its derivatives)-sialic acid conjugates, and their inhibitory activities against influenza virus sialidases and influenza virus multiplication in MDCK host cells. Deoxoglycyrrhetol-sialic acid conjugates (6d and 6e) and oleanolic acid-sialic acid conjugates (7d and 7e) showed strong inhibitory activities against three subtypes of influenza virus sialidases. These four compounds (6d, 6e, 7d and 7e) showed clear inhibition to influenza virus multiplication but not to MDCK host cell survival.