Functions and Biosynthesis of O-Acetylated Sialic Acids.

Sialic acids have a pivotal functional impact in many biological interactions such as virus attachment, cellular adhesion, regulation of proliferation, and apoptosis. A common modification of sialic acids is O-acetylation. O-Acetylated sialic acids occur in bacteria and parasites and are also receptor determinants for a number of viruses. Moreover, they have important functions in embryogenesis, development, and immunological processes. O-Acetylated sialic acids represent cancer markers, as shown for acute lymphoblastic leukemia, and they are known to play significant roles in the regulation of ganglioside-mediated apoptosis. Expression of O-acetylated sialoglycans is regulated by sialic acid-specific O-acetyltransferases and O-acetylesterases. Recent developments in the identification of the enigmatic sialic acid-specific O-acetyltransferase are discussed.

Mobilization of lymphoblasts from bone marrow to peripheral blood in childhood acute lymphoblastic leukaemia: role of 9-O-acetylated sialoglycoproteins.

Childhood acute lymphoblastic leukaemia is characterized by aberrant proliferation and accumulation of malignant lymphoblasts in bone marrow (BM), followed by their migration into circulation. An enhanced cell-surface expression of ALL-associated 9-O-acetylated sialoglycoproteins (Neu5,9Ac(2)-GPs) was demonstrated. Present investigation reports a positive correlation between the increased density of Neu5,9Ac(2)-GPs on lymphoblasts and their mobilization from BM involving enhanced Neu5,9Ac(2) on CD45 demonstrating modulation of FAK and ERK molecules. In contrast, a small population of cells, identified as haematopoietic precursors, with comparatively lesser Neu5,9Ac(2)-GPs showed increased binding towards BM stroma. Thus, Neu5,9Ac(2)-GPs is a developmentally regulated oncofoetal antigen, whose up-regulation is imperative in the interaction between lymphoblasts and BM stroma, governing their mobilization into circulation.

Positive and negative electrospray ionisation travelling wave ion mobility mass spectrometry and low-energy collision-induced dissociation of sialic acid derivatives.

Mono- or oligosaccharide-containing samples, whether they are derived from biological sources or products of chemical synthesis, are often mixtures of spatial or constitutional isomers. The possibility of characterising or performing quality control on such samples by mass spectrometry is hampered because these isomers cannot be separated by their mass-to-charge ratio alone. Therefore, the use of techniques to separate the isobaric sample compounds prior to mass spectrometric characterisation is mandatory. Travelling wave ion mobility separation offers the possibility of separating mixtures based on their compound's collisional cross-sections in the gas phase and can easily be combined with mass spectrometry for further characterisation. Here, we use 5-N-acetylneuraminic acid and several derivatives as model compounds to evaluate the separation power of travelling wave ion mobility spectrometry and present an approach to clearly identify constitutional isomers in mixtures in combination with low-energy collision-induced dissociation (CID) in the negative ion mode even if they cannot be completely separated by ion mobility.

Synthesis and inhibitory activity of sialic acid derivatives targeted at viral sialate-O-acetylesterases.

A series of sialosides modified at the 4- and 9-hydroxy group were synthesised and tested for inhibition of the viral haemagglutinin-esterase activity from various Orthomyxoviruses and Coronaviruses. While no inhibition of the sialate-4-O-acetylesterases from mouse hepatitis virus strain S or sialodacryoadenitis virus was found, a 9-O-methyl derivative displayed inhibitory activity against recombinant sialate-9-O-acetylesterase from influenza C virus.

Differentially regulated expression of 9-O-acetyl GD3 (CD60b) and 7-O-acetyl-GD3 (CD60c) during differentiation and maturation of human T and B lymphocytes.

GD3 (CD60a) and its 9-O-acetylated variant (CD60b) are intracellular regulators of apoptosis in T lymphocytes. Surface expressed 9-O-acetyl- and 7-O-acetyl-GD3 (CD60b and CD60c) may have a functional impact on activated T and B cells. In order to investigate the balance between surface and intracellular expression and synthesis and degradation of these glycosphingolipids in human lymphocytes of various differentiation stages, we analyzed (i) expression of GD3 molecules on native T and B cells and thymocytes by flow cytometry and (ii) activity and regulation of possible key enzymes for CD60a,b,c synthesis and degradation at the transcriptional level. Both, surface and cytoplasmic expression of CD60a and CD60c was highest in tonsillar T cells. In thymocytes, CD60c outweighs the other CD60 variants and was mainly found in the cytoplasm. All lymphocyte preparations contained sialate O-acetyltransferase activity producing 7-O-acetyl-GD3. Sialidase activity was highest in peripheral blood lymphocytes followed by thymocytes and tonsillar T and B cells. Transcription of GD3 synthase (ST8SiaI), the key enzyme for GD3 synthesis, was highest in tonsillar T cells, whereas transcriptional levels of sialidase NEU3 and O-acetylesterase H-Lse were lowest in activated T cells. This balance between enzymes of sialic acid metabolism may explain the strong overall staining intensity for all GD3 forms in T cells. Both CASD1, presumably encoding a sialic acid-specific O-acetyltransferase, and H-Lse showed highest transcription in peripheral B lymphocytes corresponding to the low expression of CD60b and c in these cells. Our data point to regulatory functions of these anabolic and catabolic key enzymes for the expression of GD3 and its O-acetylated variants in lymphocytes at a given differentiation stage.

Influenza C virus NS1 protein counteracts RIG-I-mediated IFN signalling.

The nonstructural proteins 1 (NS1) from influenza A and B viruses are known as the main viral factors antagonising the cellular interferon (IFN) response, inter alia by inhibiting the retinoic acid-inducible gene I (RIG-I) signalling. The cytosolic pattern-recognition receptor RIG-I senses double-stranded RNA and 5'-triphosphate RNA produced during RNA virus infections. Binding to these ligands activates RIG-I and in turn the IFN signalling. We now report that the influenza C virus NS1 protein also inhibits the RIG-I-mediated IFN signalling. Employing luciferase-reporter assays, we show that expression of NS1-C proteins of virus strains C/JJ/50 and C/JHB/1/66 considerably reduced the IFN-ß promoter activity. Mapping of the regions from NS1-C of both strains involved in IFN-ß promoter inhibition showed that the N-terminal 49 amino acids are dispensable, while the C-terminus is required for proper modulation of the IFN response. When a mutant RIG-I, which is constitutively active without ligand binding, was employed, NS1-C still inhibited the downstream signalling, indicating that IFN inhibitory properties of NS1-C are not necessarily linked to an RNA binding mechanism.

The human Cas1 protein: a sialic acid-specific O-acetyltransferase?

Sialic acids are important sugars at the reducing end of glycoproteins and glycolipids. They are among many other functions involved in cell-cell interactions, host-pathogen recognition and the regulation of serum half-life of glycoproteins. An important modification of sialic acids is O-acetylation, which can alter or mask the biological properties of the parent sialic acid molecule. The nature of mammalian sialate-O-acetyltransferases (EC 2.3.1.45) involved in their biosynthesis is still unknown. We have identified the human CasD1 (capsule structure1 domain containing 1) gene as a candidate to encode the elusive enzyme. The human CasD1 gene encodes a protein with a serine-glycine-asparagine-histidine hydrolase domain and a hydrophobic transmembrane domain. Expression of the Cas1 protein tagged with enhanced green fluorescent protein in mammalian and insect cells directed the protein to the medial and trans-cisternae of the Golgi. Overexpression of the Cas1 protein in combination with α-N-acetyl-neuraminide α-2,8-sialyltransferase 1 (GD3 synthase) resulted in an up to 40% increased biosynthesis of 7-O-acetylated ganglioside GD3. By quantitative real-time polymerase chain reaction, we found up to 5-fold increase in CasD1 mRNA in tumor cells overexpressing O-Ac-GD3. CasD1-specific small interfering RNA reduced O-acetylation in tumor cells. These results suggest that the human Cas1 protein is directly involved in O-acetylation of α2-8-linked sialic acids.

A seven plasmid-based system for the rescue of influenza C virus.

We report the establishment of a reverse-genetics system for the rescue of recombinant influenza C/JJ/50 virus from seven plasmids. The nucleotide sequence of the whole C/JJ/50 genome was determined and full-length cDNAs were cloned into an RNA pol I/pol II-based bidirectional vector. Transfection of Vero cells and subsequent amplification on MDCK cells yielded viral HA titres of 128. The utility of this bidirectional approach was proved by generating a reassortant virus encoding the NS segment from strain C/JHB/1/66 and a virus with mutations in the noncoding ends of PB1. The latter virus, which has a base-pair mutation within the proposed double-stranded region of the PB1 termini, exhibited impaired replication. In conclusion, our efficient seven-plasmid system for the rescue of recombinant influenza C virus may be used to study the influenza C virus life cycle in more detail and for generation of influenza C virus-based vectors.

Influenza C virus and bovine coronavirus esterase reveal a similar catalytic mechanism: new insights for drug discovery.

Both, the influenza C (INF-C) virus haemagglutinin esterase fusion and bovine coronavirus (BCoV) haemagglutinin esterase surface glycoproteins exhibit a lectin binding capability and a receptor-destroying 9-O-acetyl esterase activity that recognise 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac(2))-containing glycans. Here we report nuclear magnetic resonance and molecular modelling studies on the 9-O-acetyl esterase showing that the alpha-configured Neu5,9Ac(2) is strictly preferred by the INF-C and BCoV esterases. Interestingly, we have discovered that the INF-C esterase function releases acetate independently of the chemical nature of the aglycon moiety, whereas subtle differences in substrate recognition were found for BCoV esterase. Analysis of the apo and complexed X-ray crystal structure of INF-C esterase revealed that binding of 9-O-acetylated N-acetylneuraminic acids is a dynamic process that involves conformational rearrangement of serine-57 in the esterase active site. This study provides valuable insights towards the design of drugs to combat INF-C virus and coronavirus infections causing outbreaks of upper respiratory infections and severe diarrhea in calves, respectively.

Differential surface expression and possible function of 9-O- and 7-O-acetylated GD3 (CD60 b and c) during activation and apoptosis of human tonsillar B and T lymphocytes.

The disialoganglioside GD3 (CD60 a) and its O-acetylated variants have previously been described as surface molecules of human T lymphocytes of the peripheral blood system. Here we report the expression of the 9-O-, and 7-O-acetylated disialoglycans of GD3 (CD60 b and CD60 c respectively) on human tonsillar lymphocytes. CD60 b and c are surface-expressed on activated germinal centre B cells and colocalize in raft-like structures on the cell surface together with the cytoplasmic tyrosine kinase Lyn and Syk. Addition of CD60 b and c mAb together with anti-IgM/IL-4 to in vitro cultivated tonsillar B cells resulted in a costimulatory effect. During spontaneous and staurosporine-induced apoptosis a distinct population of activated annexin V+/CD60 b+/CD60 c- B cells was observed. CD60 b and c are also found on cells of the extrafollicular T cell area. On tonsillar T cells, CD60 b mAb had a costimulatory effect together with PHA while CD60 c mAb alone was sufficient to induce proliferation. In further contrast to B cells, during apoptosis a distinct CD60 b+ T cell subpopulation was not observed. Together, surface-expressed CD60 b and c are differently expressed on tonsillar B and T cells and may be involved in the regulation of activation and apoptosis of lymphocytes in secondary lymphatic tissue.

Localisation and distribution of O-acetylated N-acetylneuraminic acids, the endogenous substrates of the hemagglutinin-esterases of murine coronaviruses, in mouse tissue.

Infections by mouse hepatitis viruses result in disease of the liver, the gastrointestinal tract, respiratory tract, and the central nervous system. Coronaviruses related to mouse hepatitis virus express a hemagglutinin-esterase surface glycoprotein, which specifically hydrolyses either 5-N-acetyl-4-O-acetyl neuraminic acid (Neu4,5Ac(2)) or 5-N-acetyl-9-O-acetyl neuraminic acid (Neu5,9Ac(2)). Moreover, these sialic acids represent potential cellular receptor determinants for murine coronaviruses. Until now, the distribution of these sialic acids in mouse brain was not thoroughly investigated. Particularly Neu4,5Ac(2) was not yet found in mouse brain. Using a sensitive method of gas chromatography coupled to mass spectrometry in the electron impact mode of ionization this manuscript demonstrates the occurrence of 13 different sialic acids varying in their alkyl and acyl substituents in mouse tissues including 5-N-acetyl-4-O-acetyl-9-O-lactyl-neuraminic acid (Neu4,5Ac(2)9Lt), 5-N-acetyl-9-O-lactyl-neuraminic acid (Neu5Ac9Lt), 5-N-acetyl-8-O-methyl-neuraminic acid (Neu5Ac8Me) and the 1,7-lactone (Neu5Ac1,7L) of neuraminic acid. Neu4,5Ac(2), relatively abundant in the gut, was present as a minor compound in all tissues, including liver, olfactory lobe, telencephalon, metencephalon and hippocampus. Neu5,9Ac(2) was also found in these tissues, except in the liver. It is suggested that these sialic acids represent the endogenous substrate and receptor determinants for murine coronaviruses.

Interferon gamma promotes survival of lymphoblasts overexpressing 9-O-acetylated sialoglycoconjugates in childhood acute lymphoblastic leukaemia (ALL).

An enhanced linkage-specific 9-O-acetylated sialic acid (9-O-AcSA) on peripheral blood mononuclear cells (PBMC) of children with acute lymphoblastic leukaemia, ALL (PBMC(ALL), 9-O-AcSA+ cells) was demonstrated by using a lectin, Achatinin-H, whose lectinogenic epitope was 9-O-AcSAalpha2-6GalNAc. Our aim was to evaluate the in vitro contributory role of this glycotope (9-O-AcSAalpha2-6GalNAc) towards the survival of these 9-O-AcSA+ cells in ALL patients. For direct comparison, 9-O-AcSA- cells were generated by removing O-acetyl group of 9-O-AcSA present on PBMC(ALL) using O-acetyl esterase. An elevated level of serum interferon gamma (IFN-gamma) in affected children led us to think that PBMC(ALL) are continuously exposed specifically to this cytokine. Accordingly, 9-O-AcSA+ and 9-O-AcSA- cells were exposed in vitro to IFN-gamma. A twofold increased NO release along with inducible NO synthase (iNOS) mRNA expression by the 9-O-AcSA+ cells was observed as compared to the 9-O-AcSA- cells. The decreased viability of IFN-gamma exposed 9-O-AcSA- cells as compared to 9-O-AcSA+ cells were reflected from a 5.0-fold increased caspase-3-like activity and a 10.0-fold increased apoptosis in the 9-O-AcSA- cells when production of NO was lowered by adding competitive inhibitor of iNOS in reaction mixture. Therefore, it may be envisaged that a link exists between induction of this glycotope and their role in regulating viability of PBMC(ALL). Taken together, it is reasonable to hypothesise that O-acetylation of sialic acids on PBMC(ALL) may be an additional mechanism that promotes the survival of lymphoblasts by avoiding apoptosis via IFN-gamma-induced NO production.

Nidovirus sialate-O-acetylesterases: evolution and substrate specificity of coronaviral and toroviral receptor-destroying enzymes.

Many viruses achieve reversible attachment to sialic acid (Sia) by encoding envelope glycoproteins with receptor-binding and receptor-destroying activities. Toroviruses and group 2 coronaviruses bind to O-acetylated Sias, presumably via their spike proteins (S), whereas other glycoproteins, the hemagglutinin-esterases (HE), destroy Sia receptors by de-O-acetylation. Here, we present a comprehensive study of these enzymes. Sialate-9-O-acetylesterases specific for 5-N-acetyl-9-O-acetylneuraminic acid, described for bovine and human coronaviruses, also occur in equine coronaviruses and in porcine toroviruses. Bovine toroviruses, however, express novel sialate-9-O-acetylesterases, which prefer the di-O-acetylated substrate 5-N-acetyl-7(8),9-di-O-acetylneuraminic acid. Whereas most rodent coronaviruses express sialate-4-O-acetylesterases, the HE of murine coronavirus DVIM cleaves 9-O-acetylated Sias. Under the premise that HE specificity reflects receptor usage, we propose that two types of Sias serve as initial attachment factors for coronaviruses in mice. There are striking parallels between orthomyxo- and nidovirus biology. Reminiscent of antigenic shifts in orthomyxoviruses, rodent coronaviruses exchanged S and HE sequences through recombination to extents not appreciated before. As for orthomyxovirus reassortants, the fitness of nidovirus recombinant offspring probably depends both on antigenic properties and on compatibility of receptor-binding and receptor-destroying activities.

Recombinant viral sialate-O-acetylesterases.

Viral O-acetylesterases were first identified in several viruses, including influenza C viruses and coronaviruses. These enzymes are capable of removing cellular receptors from the surface of target cells. Hence they are also known as "receptor destroying" enzymes. We have cloned and expressed several recombinant viral O-acetylesterases. These enzymes were secreted from Sf9 insect cells as chimeric proteins fused to eGFP. A purification scheme to isolate the recombinant O-acetylesterase of influenza C virus was developed. The recombinant enzymes derived from influenza C viruses specifically hydrolyze 9-O-acetylated sialic acids, while that of sialodacryoadenitis virus, a rat coronavirus related to mouse hepatitis virus, is specific for 4-O-acetylated sialic acid. The recombinant esterases were shown to specifically de-O-acetylate sialic acids on glycoconjugates. We have also expressed esterase knockout proteins of the influenza C virus hemagglutinin-esterase. The recombinant viral proteins can be used to unambiguously identify O-acetylated acids in a variety of assays.

Infectious salmon anemia virus specifically binds to and hydrolyzes 4-O-acetylated sialic acids.

Infectious salmon anemia virus (ISAV) is the causative agent of infections in farmed Atlantic salmon. ISAV presumably represents a new genus within the Orthomyxoviridae. ISAV has been shown earlier to exhibit a receptor-destroying activity, which was defined as an acetylesterase with unknown specificity. We have analyzed the substrate specificity of the ISAV esterase in detail. Purified ISAV hydrolyzed free 5-N-acetyl-4-O-acetyl neuraminic acid. In addition, the purified 9-O-acetylated sialic acid derivative was also hydrolyzed, but at lower rates. When we used a glycosidically bound substrate, ISAV was unable to hydrolyze 9-O-acetylated sialic acid, which represents the major substrate for the influenza C virus esterase. ISAV completely de-O-acetylated glycoprotein-bound 5-N-acetyl-4-O-acetyl neuraminic acid. Thus, the enzymatic activity of the hemagglutinin-esterase of ISAV is comparable to that of the sialate-4-O-esterases of murine coronaviruses and related group 2 coronaviruses. In addition, we found that ISAV specifically binds to glycoproteins containing 4-O-acetylated sialic acids. Both the ISAV esterase and recombinant rat coronavirus esterase specific for 4-O-acetylated sialic acids hydrolyzed ISAV receptors on horse and rabbit erythrocytes, indicating that this sialic acid represents a receptor determinant for ISAV.

Identification and characterization of viral structural proteins of infectious salmon anemia virus.

Infectious salmon anemia virus (ISAV) is an unclassified Orthomyxovirus that has been shown to contain a segmented genome with eight single-stranded RNA species coding for 10 viral proteins. Four major structural proteins were characterized in the present study: two glycosylated proteins with estimated molecular masses of 42 and 50 kDa, one 66-kDa phosphoprotein, and one 22-kDa protein. Examination of lysed virions revealed the two glycoproteins and the 22-kDa protein in the soluble fraction, while the 66-kDa phosphoprotein and a minor part of the 22-kDa protein were found in the pelleted fraction. Immunofluorescence staining of infected cells demonstrated that the 22-kDa protein was a late protein accumulating in the nucleus. We conclude that the 66-kDa protein is the nucleoprotein, the 22-kDa protein is the matrix protein, and the 42- and 50-kDa proteins are the surface proteins. Radioimmunoprecipitation analysis of the 42-kDa glycoprotein, which was previously shown to represent the ISAV hemagglutinin, indicated that this protein exists at least as dimers. Further, by labeling of purified ISAV with [1,3-(3)H]diisopropyl fluorophosphate, it was also demonstrated that the viral esterase is located with the hemagglutinin. This finding was confirmed by demonstration of acetylesterase activity in affinity-purified hemagglutinin preparations. Finally, the active-site serine residue could be tentatively identified at position 32 within the amino acid sequence of the hemagglutinin of ISAV strain Glesvaer/2/90. It is proposed that the ISAV vp66 protein be termed nucleoprotein, the gp42 protein be termed HE protein, and the vp22 protein be termed matrix protein.

Common RNA replication signals exist among group 2 coronaviruses: evidence for in vivo recombination between animal and human coronavirus molecules.

5' and 3' UTR sequences on the coronavirus genome are known to carry cis-acting elements for DI RNA replication and presumably also virus genome replication. 5' UTR-adjacent coding sequences are also thought to harbor cis-acting elements. Here we have determined the 5' UTR and adjacent 289-nt sequences, and 3' UTR sequences, for six group 2 coronaviruses and have compared them to each other and to three previously reported group 2 members. Extensive regions of highly similar UTR sequences were found but small regions of divergence were also found indicating group 2 coronaviruses could be subdivided into those that are bovine coronavirus (BCoV)-like (BCoV, human respiratory coronavirus-OC43, human enteric coronavirus, porcine hemagglutinating encephalomyelitis virus, and equine coronavirus) and those that are murine hepatitis virus (MHV)-like (A59, 2, and JHM strains of MHV, puffinosis virus, and rat sialodacryoadenitis virus). The 3' UTRs of BCoV and MHV have been previously shown to be interchangeable. Here, a reporter-containing BCoV DI RNA was shown to be replicated by all five BCoV-like helper viruses and by MHV-H2 (a human cell-adapted MHV strain), a representative of the MHV-like subgroup, demonstrating group 2 common 5' and 3' replication signaling elements. BCoV DI RNA, furthermore, acquired the leader of HCoV-OC43 by leader switching, demonstrating for the first time in vivo recombination between animal and human coronavirus molecules. These results indicate that common replication signaling elements exist among group 2 coronaviruses despite a two-cluster pattern within the group and imply there could exist a high potential for recombination among group members.

Identification and characterization of adsorbed serum sialoglycans on Leishmania donovani promastigotes.

Sialic acids as terminal residues of oligosaccharide chains play a crucial role in several cellular recognition events. The presence of sialic acid on promastigotes of Leishmania donovani, the causative organism of Indian visceral leishmaniasis, was demonstrated by fluorimetric high-performance liquid chromatography showing Neu5Ac and, to a minor extent, Neu5,9Ac2. The presence of Neu5Ac was confirmed by GC/MS analysis. Furthermore, binding with sialic acid-binding lectins Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin (MAA), and Siglecs showed the presence of both alpha2,3- and alpha2,6-linked sialic acids. No endogenous biosynthetic machinery for Neu5Ac could be demonstrated in the parasite. Concomitant western blotting of parasite membranes and culture medium with SNA demonstrated the presence of common sialoglyconjugates (123, 90, and 70 kDa). Similarly, binding of MAA with parasite membrane and culture medium showed three analogous sialoglycans corresponding to 130, 117, and 70 kDa, indicating that alpha2,3- and alpha2,6-linked sialoglycans are adsorbed from the fetal calf serum present in the culture medium. L. donovani promastigotes also reacted with Achatinin-H, a lectin that preferentially identifies 9-O-acetylated sialic acid in alpha2-->6 GalNAc linkage. This determinant was evidenced on parasite cell surfaces by cell agglutination, ELISA, and flow cytometry, where its binding was abolished by pretreatment of cells with a recombinant 9-O-acetylesterase derived from the HE1 region of the influenza C esterase gene. Additionally, binding of CD60b, a 9-O-acetyl GD3-specific monoclonal antibody, corroborated the presence of terminal 9-O-acetylated disialoglycans. Our results indicate that sialic acids (alpha2-->6 and alpha2-->3 linked) and 9-O-acetyl derivatives constitute components of the parasite cell surface.

The sialate-4-O-acetylesterases of coronaviruses related to mouse hepatitis virus: a proposal to reorganize group 2 Coronaviridae.

Group 2 coronaviruses are characterized within the order Nidovirales by a unique genome organization. A characteristic feature of group 2 coronaviruses is the presence of a gene encoding the haemagglutinin-esterase (HE) protein, which is absent in coronaviruses of groups 1 and 3. At least three coronavirus strains within group 2 expressed a structural protein with sialate-4-O-acetylesterase activity, distinguishing them from other members of group 2, which encode an enzyme specific for 5-N-acetyl-9-O-acetylneuraminic acid. The esterases of mouse hepatitis virus (MHV) strains S and JHM and puffinosis virus (PV) specifically hydrolysed 5-N-acetyl-4-O-acetylneuraminic acid (Neu4,5Ac2) as well as the synthetic substrates p-nitrophenyl acetate, 4-methylumbelliferyl acetate and fluorescein diacetate. The K(m) values of the MHV-like esterases for the latter substrates were two- to tenfold lower than those of the sialate-9-O-acetylesterases of influenza C viruses. Another unspecific esterase substrate, alpha-naphthyl acetate, was used for the in situ detection of the dimeric HE proteins in SDS-polyacrylamide gels. MHV-S, MHV-JHM and PV bound to horse serum glycoproteins containing Neu4,5Ac2. De-O-acetylation of the glycoproteins by alkaline treatment or incubation with the viral esterases resulted in a complete loss of recognition, indicating a specific interaction of MHV-like coronaviruses with Neu4,5Ac2. Combined with evidence for distinct phylogenetic lineages of group 2 coronaviruses, subdivision into subgroups 2a (MHV-like viruses) and 2b (bovine coronavirus-like viruses) is suggested.