The HE protein of MHV is encoded by a gene located immediately upstream of the spike gene. It is expressed from mRNA 2-1; the molecular mass is approximately 60 to 69 kDa. In virions, it is found as a dimer anchored in the viral membrane by a C-terminal transmembrane region. Expression of the HE gene is highly variable between MHV strains. Functional HE proteins have been detected in MHV-JHM (
27), MHV-S (
38), and MHV-DVIM (
31,
32). In MHV-S, large levels of the HE protein are found, while MHV-JHM expresses relatively low amounts (
27,
38). Different MHV-JHM isolates express variable levels of HE, depending on the number of UCUAA repeats at the 3′ end of the leader RNA (
18).
The presence of HE is not strictly required for MHV replication. MHV-A59 and several other MHV strains do not express HE (
17,
27,
38). In MHV-A59, it is not expressed due to a missing initiation codon (
17). In addition, the upstream promoter determining synthesis of mRNA 2-1 is destroyed in this strain. In other MHV strains, mutations and deletions at the 3′ end of the HE gene have been detected; as a result, HE proteins without a transmembrane anchor are encoded. Biosynthesis of such truncated forms could be detected neither in lysates of infected cells nor in culture supernatants (
38). Interaction of HE alone with target cells is apparently not sufficient for infectivity. MHV-DVIM replication is inhibited by a monoclonal antibody specific for the MHV receptor, indicating that interaction of the viral spike protein with cellular receptor molecules is mandatory for infection (
6). The presence of HE may, however, modulate tissue tropism particularly within the central nervous system. MHV strains expressing an HE protein exhibit some preference for infecting neurons (for a review, see reference
1). Differences in neuropathogenicity of viruses with or without HE expression are at least partially derived from immune responses against HE. Passive immunization of mice with HE-specific monoclonal antibodies resulted in protection from a lethal infection, possibly by inhibition of virus spread through the central nervous system (
39). MHV variants with mutations in the HE gene were isolated from such animals at late stages of infection (
41). In a recent study, mice were infected with a chimeric MHV-A59 strain containing an HE protein derived from cells transfected with a defective interfering vector expressing the HE gene of MHV-JHM. Data obtained in this study indicated an enhanced early innate response caused by transient expression of HE (
42).
In addition to MHV strains, several other viruses have been shown to express HE proteins. Among these, BCV and influenza C virus have been studied most extensively. HE proteins of these viruses are receptor-destroying enzymes, removing 9-
O-acetyl groups from sialic acid-containing cellular receptor glycoproteins (
11,
23,
25,
34,
35). In contrast, data on substrate specificities of MHV esterases are limited. Enzymatic activity was mostly determined with
p-nitrophenylacetate (
pNPA) as the substrate (
6,
21,
40). Recently, the esterase of MHV-DVIM was found to remove acetyl groups from the natural substrate bovine mandibulary gland mucin (BSM) at very low levels (
33).
We recently characterized the HE protein of puffinosis virus (PV), a coronavirus closely related to MHV (
14). In that study, we compared substrate specificities of PV and influenza C virus. Results obtained from this comparison led us to propose that compounds different from 5-
N-acetyl-9-
O-acetyl sialic acid (Neu5,9Ac
2) may be natural substrates for the PV HE. Because of the high amino acid sequence similarity between the HE proteins of PV and MHV, we have now extended our investigation on the substrate specificity of the MHV esterase. In this report, we provide evidence that 4-O-acetylated sialic acid (Neu4,5Ac
2), but not Neu5,9Ac
2, is a natural substrate for the HE protein of MHV-S.
DISCUSSION
In this study, we investigated the substrate specificity of the HE protein of MHV-S. Other viruses known to express evolutionarily related proteins are influenza C viruses (
5,
11,
34), BCV (
35,
36), human coronavirus OC43 (
12,
43), hemagglutinating encephalomyelitis virus (
26), and bovine torovirus (
2). Several of these viral esterases have been characterized in terms of their substrate specificities and in all instances tested have been shown to recognize 9-O-acetylated sialic acids. Enzymatic activities of HE proteins in MCV strains have been described, but few data on their substrates and binding activities have been published (for a review, see reference
1). Recently, more data became available. First, Sugiyama et al. (
33) reported significant differences on cleavage of a natural substrate known to contain high amounts of O-acetylated sialic acids. They found that MHV-DVIM, the only MHV strain exhibiting hemagglutinating activity, can hydrolyze O-acetylated sialic acids present on the natural substrate BSM, but at limited rates compared to other viral esterases. Furthermore, data published in this work indicated that MHV-S was essentially unable to liberate acetic acid from BSM (
33). We have recently investigated another coronavirus, PV, and found similar differences regarding acetate release from Neu5,9Ac
2. Particularly, BSM was found to be no substrate for PV, a virus closely related to MHV (
14). These data had prompted us to hypothesize that other, unidentified O-acetylated compounds may be substrates for PV and closely related coronaviruses.
In this study we used MHV-S, a strain expressing high levels of HE protein (
38). In contrast to BCV and influenza C virus, MHV-S exhibited no esterase activity with BSM and was in addition unable to remove influenza C virus receptors from erythrocytes. To clarify the reasons for these differences, we wanted to gain further information on the enzymatic activity of the MHV-S HE protein. We used either chemically synthesized sialic acid derivatives or sialic acids prepared from BSM, ESM, or guinea pig serum glycoproteins to characterize substrate specificities of the MHV-S esterase. We identified Neu4,5Ac
2 as the only sialic acid derivative hydrolyzed by MHV-S. Other sialic acids with O-acetylation on the glycerol side chain were not de-O-acetylated at detectable amounts. MHV-S was able to hydrolyze acetyl esters from free as well as glycosidically linked Neu4,5Ac
2. In addition, we have demonstrated that this novel substrate specificity of MHV-S is a property of the viral HE protein. HE expressed by recombinant vaccinia virus exhibited the same reactivity with Neu4,5Ac
2 as observed with MHV-S.
Recently, Sugiyama et al. reported acetate release by MHV-DVIM from isolated murine brush border membranes (
33). However, in the case of MHV-DVIM, it remains to be determined whether this MHV strain also exhibits 4-
O-acetylesterase or the more classical 9-
O-acetylesterase activity. Taking into consideration the close relationship between amino acid sequences of MHV esterases, it appears likely that all MHV HE proteins are specific for Neu4,5Ac
2. On the other hand, the exclusive specificity observed for the HE protein of MHV-S may be the result of subtle changes in the three-dimensional configuration of the viral enzyme during evolution. Possibly there exist viral esterases that recognize
O-acetyl esters on sialic acids in positions 4 and 9. The possibility arises that in addition to MHV, other viruses with an esterase specific for Neu4,5Ac
2 exist, infecting particularly animals which are known to possess such sialic acid derivatives, e.g., horses or guinea pigs (
13). However, to our knowledge there is no evidence that MHV-S itself causes infections in these animals. It will be interesting to test specificities of other coronaviruses with HE proteins.
Binding assays revealed that MHV-S exhibits a concentration-dependent affinity to glycoproteins with Neu4,5Ac2. We provide two forms of evidence that 4-O-acetylation is required for binding. First, saponification of O-acetyl groups on guinea pig and horse serum glycoproteins resulted in a complete loss of affinity. Second, BSM, which possesses sialic acids O-acetylated in the glycerol side chain but not in position 4, was not a binding substrate for MHV-S. Thus, one may speculate that 4-O-acetylated sialic acids on glycoconjugates at the surface of cells can serve as additional viral receptors.
Current evidence suggests that infection by MHV strictly depends on the interaction of the viral spike protein with the MHV receptor present at the surface of target cells (
6). This was concluded from experiments designed to infect cells expressing influenza C virus receptors containing Neu5,9Ac
2. Such cells were not infected by MHV-DVIM unless the MHV receptor was expressed from the transfected gene. Since we now provide evidence that influenza C virus receptors are not bound by the HE protein of MHV-S, it remains to be determined if this also applies to MHV-DVIM. In the future, we will test whether MHV-S can infect cells expressing Neu4,5Ac
2but lacking the MHV receptor. Such experiments may shed light on whether the presence of the MHV receptor is a prerequisite for infection by MHV-S. Neu4,5Ac
2 may either serve as secondary receptor modulating tissue tropism of HE-expressing MHV strains or represent an alternative receptor facilitating infection of cells devoid of the MHV receptor.
Since Neu4,5Ac
2 has not yet been found in mice (
13), the question arises about potential substrates for the HE protein in this host. In further experiments, it may be rewarding to explore the binding activity of recombinant, soluble MHV-S HE with 4-O-acetylated sialic acid-bearing glycoconjugates. This may be a useful tool for the histochemical detection of Neu4,5Ac
2 in mice. Similar approaches to detect Neu5,9Ac
2 have been described for recombinant, soluble influenza C virus HE (
15,
16) as well as for purified influenza C virus (
10,
44,
45).
In summary, we have identified a viral enzyme exhibiting a previously unidentified specificity. In addition to the sialidases of influenza A and B viruses and paramyxoviruses and the sialate-9-
O-acetylesterases of influenza C and BCV, a third type of receptor-destroying enzyme specifically cleaving 4-
O-acetyl groups, has now been identified (Fig.
6).