https://onlinelibrary.wiley.com/doi/10.1002/bies.202000312
Biocidal agents such as formaldehyde and glutaraldehyde are able to inactivate several coronaviruses including SARS‐CoV‐2. In this article, an insight into one mechanism for the inactivation of these viruses by those two agents is presented, based on analysis of previous observations during electron microscopic examination of several members of the orthocoronavirinae subfamily, including the new virus SARS‐CoV‐2. This inactivation is proposed to occur through Schiff base reaction‐induced conformational changes in the spike glycoprotein leading to its disruption or breakage, which can prevent binding of the virus to cellular receptors. Also, a new prophylactic and therapeutic measure against SARS‐CoV‐2 using acetoacetate is proposed, suggesting that it could similarly break the viral spike through Schiff base reaction with lysines of the spike protein. This measure needs to be confirmed experimentally before consideration. In addition, a new line of research is proposed to help find a broad‐spectrum antivirus against several members of this subfamily.
Coronaviridae is a large family of enveloped single‐stranded RNA viruses. It is divided into two subfamilies: orthocoronavirinae and letovirinae.[1] Orthocoronavirinae consists of four genera named Alpha, Beta, Delta, and Gamma coronaviruses. Betacoronaviruses include several viruses that infect humans causing life‐threatening diseases such as SARS‐CoV, SARS‐CoV‐2 and Middle East respiratory syndrome coronavirus (MERS‐CoV).
Coronaviruses have a set of structural proteins, one of them is the spike “S” protein. This protein is a class I virus fusion protein that forms homotrimers giving clove‐shaped molecules protruding from the virus surface.[2, 3] These viruses use this protein to attach to host cell receptors to allow cellular entry. Each spike monomer is composed of two subunits; S1 subunit which harbors the receptor‐binding domain (RBD) and S2 subunit which is required for the fusion of cell and virus membranes. S protein is cleaved at a site between S1 and S2 either during synthesis or during cellular entry to give the two subunits.[2, 3] In the case of SARS‐CoV‐2, S is cleaved during synthesis at the S1/S2 site (Figure 1A) by furin[4, 5] and the two subunits remain non‐covalently bound. This protein is further cleaved during cellular entry at another site termed S2′ by the transmembrane serine protease 2 (TMPRSS2)[4] to release the fusion peptide (FP) to initiate membrane fusion. Both cleavage events are required for successful infection by SARS‐CoV‐2. Following receptor binding, S1 subunit separates from the virus particle and the S2 subunit undergoes conformational changes ultimately bringing viral and cellular membranes closer to each other and driving their fusion to release the viral genetic material inside the cell.[2]
FIGURE 1 Open in figure viewer PowerPoint Domains (A) and amino acid sequence (B) of S protein of SARS‐CoV‐2 showing location of lysine residues (in red)
Each subunit of the spike monomers of SARS‐CoV‐2 consists of several regions (Figure 1A). The S1 subunit (residues 14–685) consists of an N‐terminal domain (NTD) and a C‐terminal domain (CTD), while the S2 subunit (residues 686–1273) includes an FP, heptapeptide repeat sequence 1,2 (HR1, HR2), transmembrane domain (TM) and cytoplasmic tail (CT).[6] The CTD acts as the receptor binding domain (RBD) for this virus,[7] in which the receptor binding motif (RBM) is the site for receptor binding. S of SARS‐CoV‐2 is a dynamic structure[8] in which the RBDs are able to transit between down and up conformations. A recent analysis of the stability of the S protein in the absence of receptors reported that spikes with RBDs in the up conformation are less stable than those with RBDs in the down conformation[9] angiotensin‐converting enzyme 2 (hACE2) receptors can only bind to RBDs in the up conformation, and this binding is suggested to help lock the RBDs in this unstable conformation until S1 monomers dissociation.[10]
S protein of SARS‐CoV‐2 is composed of 1273 amino acids, 61 of which are lysine residues (Figure 1B). These lysines are almost equally distributed between the two subunits; 30 in S1 and 31 in S2. Some of them appear to help stabilize the RBD in the down‐conformation and hence stabilize the whole S protein.[9]
Glutaraldehyde is a dialdehyde commonly used in laboratory research as a chemical fixative.[11] It has the chemical formula C5H8O2 with two carbonyl groups (one at each end). Formaldehyde is another aldehyde with only one carbonyl group and is also used in chemical fixation and virus inactivation.[12] It has the chemical formula CH2O and it can polymerize to form paraformaldehyde. Both agents have the ability to inactivate several types of coronaviruses.[13] By analyzing observations from electron microscopic examination of several coronaviruses, a mechanism for this inactivation process, via interaction with viral spikes, is proposed. Also, an intervention against SARS‐CoV‐2 is proposed, using ketone bodies (in particular acetoacetate) which circulate in the human blood and interstitial fluids, suggesting that they could inactivate the extracellular virions in a similar pattern.
In one study,[14] during electron microscopic examination of Vero E6 cells infected with SARS‐CoV, Snijder et al. reported that the viral spikes of secreted extracellular virions were only visible in cryofixed samples but were rarely seen in chemically fixed samples (with 1.5% glutaraldehyde). This loss of viral spikes was also observed in the chemically‐fixed (with 2% glutaraldehyde) extracellular virions of the transmissible gastroenteritis coronavirus (genus Alphacoronavirus), which affects newborn piglets.[15] For the new virus SARS‐CoV‐2, virus particles in the supernatant of infected cell cultures that were inactivated with 2% paraformaldehyde also showed significant loss of viral spikes.[16] Also, SARS‐CoV‐2 progeny virions in glutaraldehyde‐fixed samples were reported to have few or no apparent spikes.[17]
Chemical fixation of samples is known to cause the cessation of all biological processes inside cells, including the secretion of new virions from infected cells. This in turn eliminates the possibility of the disruption of the virus assembly process or the pre‐mature secretion of un‐assembled virions from infected cells by the action of the fixatives as an explanation for the observed effect. Therefore, this effect of chemical fixation on viral spikes most probably results from direct chemical interaction between the fixative and the spike protein. The fixatives appear to interact with the spike protein in such a way that leads to its separation from the virion. Also, since this effect is evident in several members of the orthocoronavirinae subfamily, these fixatives appear to interact with a conserved sequence of the spike protein in those members.
The next question to ask is, what is the nature of this interaction? Several studies showed that glutaraldehyde's interaction with proteins occurs predominantly with lysine amino acids on the surface of proteins.[18-20] The carbonyl group of glutaraldehyde reacts with the ϵ‐amino group of lysine side chain to form a Schiff base. Schiff base reaction involves the formation of a carbon‐nitrogen double bond when the carbonyl group of an aldehyde or ketone reacts with a primary amine (Figure 2).[21] Formaldehyde was also shown to form Schiff bases with lysine side chains during interaction with proteins (Figure 3).[22] Since both fixatives share in common the ability to form Schiff bases with proteins, these bases are most likely responsible for the loss of viral spikes in the mentioned studies.[14-17] One possible explanation is that the formation of these bases with lysine residues might alter the conformation of the spike protein leading to its separation from the virion (via separation of S1 subunit from S2 subunit) (Figure 4) or alternatively, leading to its bending. In fact, there are several observations to support this view. Firstly, glutaraldehyde was shown to induce gross conformational changes in several soluble and membrane proteins through interaction with lysine residues via Schiff base reaction[20, 23] and thus, is expected to do the same in the spike protein. Similarly, formaldehyde was shown to be able to induce protein conformational changes as well.[24, 25] In the majority of these tested proteins, formaldehyde and glutaraldehyde tended to reduce the α‐helix content, which in turn loosens the protein skeleton.[25] Secondly, conformational changes induced in the spike glycoprotein of the mouse hepatitis coronavirus (MHV) A59 (genus Betacoronavirus) by other means (pH changes) could dissociate the S1 subunit from the virions and abrogate virus infectivity.[26] Similarly, conformational changes induced in the spike protein of SARS‐CoV‐2 upon binding to ACE2 receptors were followed by the dissociation of S1 monomers.[10] S1 shedding following receptor binding was also observed in the cleaved S of SARS‐CoV.[27] This suggests the wide susceptibility for S1 separation following certain conformational changes induced in the cleaved spike by different means. These changes might disrupt the interactions between the two subunits ultimately driving S1 dissociation. Indeed, conformational changes induced in the spike of MHV upon binding to receptors were reported to drive the movement of S1 away from S2 and decrease the interface between them[28] which could reduce the interactions between them and facilitate S1 shedding. Similarly, in SARS‐CoV‐2, conformational changes in ACE2‐bound spikes were shown to involve a decrease in the contact area between S1 and S2 with a reported loss of interactions between them, compared with the unbound spikes.[10] It is thus conceivable to predict that conformational changes induced by the biocidal agents can do the same, namely disrupt the interactions between S1 and S2 leading to the dissociation of S1 from the virions. This may explain one mechanism by which formaldehyde and glutaraldehyde can inactivate coronaviruses (Figure 4). Based on this, other short aldehydes and ketones that are biologically‐competent and have carbonyl groups capable of forming Schiff bases with the spike protein and altering its conformation could be speculated to break the viral spikes as well and thus, have a therapeutic potential. One such compound could be the ketone bodies. Similar to glutaraldehyde and formaldehyde, ketone bodies can also alter the conformation of proteins they react with, via Schiff base reaction.[29]
Schematic representation of the proposed SARS‐CoV‐2 spike inactivation by biocidal agents (such as formaldehyde) and by ketone bodies. Inset: representation of the spike protein with all RBDs in down or up conformation. Top: formaldehyde molecules react with lysine residues of the spike protein via Schiff base reaction and alter its conformation leading to the separation of S1 subunit from S2 subunit (which remains attached to the virion). Bottom: Ketone bodies (acetoacetate) react with the same residues of the spike protein and also alter its conformation causing the separation of S1 subunit from S2 subunit. RBDs are shown in up conformation after both reactions
There are three types of ketone bodies in the human blood.[30] These include beta‐hydroxybutyrate (C4H7O3−), acetoacetate (C4H5O3−) and the least abundant one, acetone (C3H6O). They are synthesized in the liver from fatty acids and released in the blood to act as a source of energy when there is a shortage of carbohydrate supply to the body. Thus, the level of ketone bodies increases in the plasma during periods of fasting and starvation. In fact, there are observations strongly suggesting that acetoacetate can form Schiff bases when reacting with lysine residues of proteins (Figure 5) and can alter their secondary structure,[29] similar to the effect of the mentioned fixatives. More specifically, acetoacetate reduced the α‐helix content of the tested protein, an effect similarly induced by formaldehyde and glutaraldehyde.[23-25] Moreover, ketone bodies are short compounds (three or four carbons long) and therefore should have accessibility to amino acids within proteins comparable to the short fixatives (formaldehyde and glutaraldehyde have one carbon and five carbons, respectively). Hence, ketone bodies are expected to effectively react with the viral spikes and break or bend them similar to the fixatives (Figure 4); however, this needs to be confirmed experimentally before consideration. The induction of ketosis would then have a therapeutic as well as a prophylactic potential. In infected patients, the loss of viral spikes after virions secretion from infected cells would inhibit or slow down the spread of infection to other cells and tissues in the body. Also, virus particles in body secretions will have low or abrogated infectivity toward other persons, thus rendering the infected patients relatively non‐infectious. In the non‐infected individuals, the presence of high titer of ketone bodies in the blood would decrease the magnitude of subsequent infection or even make them highly immune to infection.
