4. Relationship between C4-O- and C9-O-Acetyl SA Preferences of CoVs in Host Cell Recognition

4.1. General O-Acetylation of SA
SAs have various derivatives of more than 50 chemically different structures formed from the basic N-acetylneuraminic acid (Neu5Ac) on the main ring of pyranose and the glycerol side chain. SA are modified by acetyl-, lactyl-, methyl- and sulfo-groups individually or in multiple combinations [28]. Multiple enzymes are involved in the modifications [29]. Historically, the first discovered SA was crystallized by Gunner Blix via a hot mild acid extraction of bovine submaxillary mucin in 1936. It consisted of two acetyl groups. Among these, only one acetyl group was attached to nitrogen [30]. Blix isolated a 9-O-acetyl SA of the common SA N-acetylneuraminic acid (Neu5Ac), chemically described as 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac2). Neu5,9Ac2, Neu5Ac and Neu5Gc are naturally occurring SA species in mammals. A common modification is O-acetylation. In fact, O-acetylation of SAs is common in organisms.
The O-acetyl modification occurs in single positions of C-4, C-7, C-8 and C-9 of SA as well as in combined C-positions to yield Neu4,5Ac2, Neu5,9Ac2 and Neu5,7,9Ac3 SAs. Neu5Ac9NAc is a chemical and biologic mimic of Neu5,9Ac2 in the SA-glycans. The C-7 and/or C-9 O-acetylations are catalyzed by the SA O-acetyltransferase enzyme, Cas1 domain containing 1 (CasD1) (Figure 6). CasD1 catalyzes the addition of acetyl groups to the SA C-7 at the late Golgi apparatus compartment [31]. Thereafter, an enzyme termed “migrase” transfers the additional acetyl-group from C-7 to C-9, although this enzyme has not been identified [29]. CasD1 uses acetyl-coenzyme A as a donor substrate and CMP-Neu5Ac as an acceptor substrate, but with weak activity on CMP-Neu5Gc.
Biologically, the SA O-acetylation event confers merits to hosts such as protection from pathogenic invasion and maintenance of systemic self-homeostasis. The O-acetylation event of SAs protects the SA-containing glycans from neuraminidase (NA)/sialidase action, because O-acetyl-groups inhibit microbial NA activity. The chemical structure of the O-acetyl group is quite unstable and susceptible to esterase enzymes. Sialic acid cleavage of the di-acetylated Neu5,7,9Ac3 by bacterial NAs decreases two-fold, when compared to mono-O-acetylated Neu5Ac. The O-acetylated glycan modification invites interaction with viruses, antibodies and mammalian lectins [32]. Therefore, the SA O-acetylation modification confers specific functions to organisms.

4.2. Evolutionary Acquisition of C4-O-Acetyl and C9-O-Acetyl SA Recognition by HE Enzymes

4.2.1. C4-O-Acetyl Modification
For example, in the horse, C4-O-acetyl modification of Neu5Ac (SA) occupies more than 50% of the total SA content. The C4-O-acetylated Neu5Ac, Neu4,5Ac2, inhibits the influenza A2 virus HA. De-acetylation reagents such as NaOH or NaIO4 treatment completely hemagglutinate Neu4,5Ac2 by elimination of the C4-O-acetyl group [33]. The C4-O-acetyl Neu5Ac species are found in various sources such as equine erythrocyte GM3, starfish A. rubens and fish [34,35,36,37,38]. C4-O-acetylated Neu5Ac facilitates the initial attachment of viruses to target cells. Like the influenza C virus, infectious salmon anemia virus (ISAV), a member of the Orthomyxoviridae family, contains HE and HEF proteins to mediate virus entry and exit. C4-O-Ac Neu5Ac is the major receptor determinant of ISAV in receptor binding and destruction [38], while the influenza C virus recognizes C9-O-Ac Neu5Ac. The acetylesterase RDE of ISAV cleaves C4-O-Ac via 4-SA-O-acetylesterase with a short turnover time, whereas C9-O-Ac Neu5Ac is cleaved by 9-SA-O-acetylesterase with a long turnover time [34].
The position of SA O-acetylation is linked to functions including substrate differentiation of enzymes such as NAs and esterase by C4 O-Ac. Previous development of O-Ac site-selective NA inhibitors were based on the conceptual consideration of different O-Ac positions. The O-Ac of SAs is site-specific, as C4 of Neu5Ac is considered to be a potential position for modification. Historically, inhibitors of influenza A and B viruses-sialidases were designed by Von-Itzstein in 1993 [39]. The Ac group-based C4 substitution interacts with amino acid Glu-119 present in the active site of sialidase. Guanidine-attached C4 of C2–C3 unsaturated SA (Neu5Ac2en) inhibits activity of sialidases isolated from influenza A virus (Singapore/1/57) and B virus (Victoria/102/85). The same scenario was applied for sialidase inhibition of the human parainfluenza virus type 3, which has HN and fusion proteins [40]. The C4 of Neu5Ac2en was substituted by alkyl groups such as the O-ethyl group. For example, Zanamivir has a substitution with a 4-guanidino group with an IC50 of 25 μM. Thus, sialidase inhibition is important for C4 modification of Neu5Ac2en. Later, oseltamivir with the tradename Tamiflu (Basel, Switzerland) and zanamivir with the tradename Relenza (London, UK) were established [41]. These drugs exhibit some adverse side effects that restrict clinical use.

4.2.2. SA C9-O-Acetyl Modification
The SA 9-O-acetylation in hosts allows hosts to evade influenza A virus hemagglutinin (HA) recognition and some lectins of factor H (FH), CD22/Siglec-2 and sialoadhesin/Siglec-1. Instead, the influenza C virus HA recognizes the hosts. β-elimination and permethylation eliminate the 9-O-acetyl group from SAs. Chemical modification of the C-9 position of Neu5,9Ac2 generates a 9-N-acetyl analog, 9-acetamido-9-deoxy-N-acetylneuraminic acid (Neu5Ac9NAc), a mimic of Neu5,9Ac2 with influenza C virus-binding capacity, which is not cleaved by the HE [42]. SA O-acetylesterase regulates the presence of 7,9-O-Ac and 9-O-Ac. SA O-acetylation and deacetylation are involved in development, cancer and immunology. SA O-acetylation alters host lectin bindings such as siglecs [29]. The presence of 9-O-Ac can also reduce the activity of NAs [43]. SA modifications regulate pathogen binding or pathogen NAs. Influenza A/B/C/D viruses use SA as their entry receptors. Influenza A and B subtypes bind to SAs via HA and NA to allow endocytosis of the virus and fusion of the viral envelope with endosomes. In contrast, influenza C and D subtypes bear only one coated glycoprotein, termed the HE fusion protein (HEF). The HEF acts as the HA and NA. HEF recognizes 9-O-acetyl SA for entry into cells, while the esterase domain removes 9-O-acetyl-groups and liberates the virus from mucus and mis-assembled virus aggregates after budding. The 9-O-Ac on cells prevents the NA activity and HA binding of the influenza A type virus [44].