Myristoylation

Linkage of myristic acid (C14:0) to the N-terminal of a glycine residue found on some viral, cellular, or bacterial proteins, is known as N-terminal myristoylation [120–123]. Several viral proteins are myristoylated including the poliovirus VP4 protein, simian immunodeficiency virus (SIV) Gag protein, human immunodeficiency virus (HIV) negative regulatory factor (Nef) protein, and the pre-S1 protein of the hepatitis B virus (HBV) [124–127]. All of these proteins contain the conserved sequence motif ₁MGxxxS/T, where ‘x’ can be any amino acid [80]. Coronavirus E proteins, along with other members of the Nidovirales order, reportedly have no myristoylation motif and it is suggested to be a feature unique only to the Arteriviridae family in the order of Nidovirales [80]. However, there appears to be no experimental evidence to support this.

Ubiquitination

Ubiquitination and its counterpart, deubiquitination, are well-characterised post-translational modifications with that serve to maintain homeostasis through the regulation of cellular protein levels and their functions [128]. Viruses can exploit this component of the host cell machinery, or even encode their own ubiquitinating/deubiquitinating enzymes to drive the viral life cycle [129]. Only SARS-CoV E has so far been reported to be ubiquitinated, although the relevance has not yet been determined. The SARS-CoV non-structural protein (nsp) 3 co-localises with E and its interaction was mediated through the N-terminal ubiquitin-like domain-1 of nsp3. Independently, a ubiquitination assay further demonstrated that E can be ubiquitinated and that its ubiquitination status inversely correlates to its stability and half-life [128, 130]. Moreover, given the late expression of SARS-CoV accessory protein 8b, Keng, Åkerström [130] suggested that it might function to modulate viral production by down-regulating E production and in doing so maintain an optimal viral titre. However, this will need to be confirmed in the context of a natural infection.

Envelope and envelope proteins

The CoV E protein is unique in that it can form homotypic interactions, which allows it to oligomerise and generate an ion-channel protein known as a viroporin [135, 136]. Biochemical evidence suggests that the ability of CoV E to form homo-oligomeric multimers is dependent on its TMD. Synthetic peptides that correspond to the SARS-CoV E TMD can form dimers, trimers, and pentamers, demonstrating the importance of the TMD in CoV E homotypic interactions [137]. This was ability to produce multimeric homo-oligomers was confirmed by expression of SARS-CoV E in Sf9 insect cells. Substituting certain hydrophobic residues in the TMD with charged residues, significantly alters the electrophoretic migration rate of E to the extent that only monomers are observed [73]. To date, not many studies have investigated which TMD residues are required for CoV E homotypic interactions. Mutation of the TMD residues asparagine 15 (N15) to alanine (N15A) and valine 25 (V25) to phenylalanine (V25F) have been found to abolish the ion channelling capability of CoV E viroporin, a structure dependent on its homopentameric conformation [75, 76, 138]. Interestingly, mutation of N15A and V25F, respectively, appear to hamper the oligomerisation of CoV E, at least to some extent. The appearance of monomers in response to V25F clearly suggests that this residues plays a more crucial role in oligomerisation, as opposed to N15A, which appears to reduce the amount of pentamers only slightly [139]. The ability of CoV E to assemble into homopentameric structures is clearly important in the formation of a functional CoV E viroporin [75, 76, 135–138, 140].

Nucleocapsid and envelope proteins

It has been shown that M and E are sufficient to drive VLP formation in many CoVs and that its production is further enhanced by the co-expression of N [42, 60, 63, 64, 141]. It is thought that E and N interact with M independently and are assembled separately into VLPs. Accordingly, it is not known whether E and N interact and, in doing so, if this interaction is what could enhance virion production. Only two studies have reported a possible interaction between E and N, one for murine MHV and the other for SARS-CoV. Tseng, Wang [142] reported an interaction between SARS-CoV E and N mediated largely through the C-terminus of both proteins. Deletion of the last C-terminal residue of E markedly reduced E and N interaction although it did not seem to significantly compromise efficient VLP production. Although the study only looked at an E-N interaction in transfected cells, it is interesting to note that Maeda, Maeda [143] already found coimmunoprecipitation of structural proteins E and N in MHV-infected cells. This suggests that there might, in fact, be a physical interaction between E and N but the reason and exact requirements for this interaction remains to be determined. More research is needed to understand this interaction and whether it offers a possible explanation as to why or how VLP production is enhanced during the co-expression of M, E, and N [42, 52].

Assembly and budding: Membrane curvature

Coronaviruses are unique among enveloped viruses in that assembly of the viral envelope occurs at the ERGIC. From there, virions bud into the lumen, navigate their way through the host secretory pathway, and ultimately egress from the cell [89, 90, 160, 161]. Although assembly of the viral envelope is coordinated by M, both M and E are required for the production and release of VLPs [51, 55, 56, 60–64, 141, 162–164]. Still, deleting the E gene from several recombinant CoVs does not halt virus production but rather cripples viral production severely or produces replication-competent but propagation-defective virions [35, 39, 40, 67, 68, 150, 165, 166]. Clearly then E is involved in the CoV assembly and release, but the exact role is not yet fully understood.

The coronaviral envelope consists predominantly of M while only a small portion of E is incorporated into the viral envelope of virions [100, 167, 168]. Extensive electron microscopy (EM) studies conducted on M from a variety of CoVs provided no indication that M is capable of inducing membrane curvature on its own [51, 169, 170]. In fact, various recombinant CoVs (rCoVs) lacking the E gene (ΔE) exhibit a strikingly aberrant morphology. When C-terminus residues of MHV E were mutated to alanine, virions became temperature sensitive and took on pinched, elongated shapes rather than the typical spherical particles observed among wild type virions [171]. Plaques of recombinant MHV-ΔE exhibited a very similar aberrant morphology, presenting as small, irregular-shaped plaques with jagged edges [39]. Cells infected with recombinant SARS-CoV-ΔE (rSARS-CoV-ΔE) contained a lower number of mature virions but exhibited a higher proportion of vesicles containing a dense, granular material. This material was proposed to be the result of the aborted viral assembly process that gave rise to immature virions [35]. Most interestingly, TGEV-ΔE-infected cells contained immature virions that were blocked from being secreted into the medium. The absence of TGEV E arrested virus trafficking and, thereby, blocking full virion maturation [40]. In comparison, the phenotype of VLPs made up of M and E are described as smooth and indistinguishable from, or resembling, wild type virions, placing this morphology in stark contrast to that observed of virions lacking E [37, 63, 64]. Clearly, even though viral assembly and production is not completely stopped in the absence of E, the aberrant morphology of ΔE-virions strongly suggests that E participates in the assembly process. Most likely then, instead of coordinating viral assembly, the function of E is rather to induce membrane curvature of the viral envelope, thereby allowing CoV particles to acquire their characteristic spherical shape and morphology.

Coronavirus-infected cells contain several different membranous structures, including double-membrane vesicles (DMVs) and convoluted membranes (CMs) [172–175]. However, little is known about exactly how these structures are formed and which viral and/or host proteins are involved in this process. Co-expression of SARS-CoV nsps 3, 4, and 6 can induce membrane rearrangements that resemble the DMVs and CMs observed in CoV-infected cells [176]. The luminal loops present in full-length nsp3 and nsp4 are essential for the formation of the replicative structures seen in SARS-CoV-infected cells [176, 177]. Moreover, the cysteine residues located in the luminal loop nsp4 appear to be particularly important in the process of ER membrane rearrangement [177]. Hagemeijer, Monastyrska [177] proposed a model in which the luminal loops located between the transmembrane regions of nsp3 and 4 interact with one another to initiate the rearrangement of ER membranes and induce membrane curvature to form DMVs (Fig. 4).

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Fig. 4

Model proposed by Hagemeijer, Monastyrska [177] for the induction of ER membrane curvature. The luminal loops of CoV nsp3 and 4 are required to initiate rearrangement of the ER membrane and produce the DMVs characteristically seen in CoV-infected cells

This underpins the importance of establishing a unanimous topology for the E protein as this model could be applied to the induction of membrane curvature by E, provided E can assume multiple topologies during an infection. Should it be demonstrated that E can take on a topology with a luminal loop, this would not be inconceivable as a possible mechanism for the induction of membrane curvature initiated by E or in which E participates. Equally, as heterotypic interactions of nsp3 and 4 are required to induce ER membrane curvature, and the expression of both M and E is required for the formation of smooth, spherical CoV VLPs, it would be interesting to see if a heterotypic interaction between M and E could drive membrane curvature by a similar mechanism [176–178]. Alternatively, no research exists on the exact purpose of the N-terminus of E. Perhaps homotypic interactions mediated by the N-termini of alternating E proteins could be responsible for inducing membrane curvature by a similar mechanism. It is also worth noting that the mutation of each of the cysteine residues located in the nsp4 luminal loop abrogated the ability of nsp4 to rearrange the ER membranes [177]. This is interesting because cysteine residues are substrates for the palmitoylation of proteins associated with membranes [113]. Perhaps this corroborates the requirement of E palmitoylation, not in assembly per se, but rather by anchoring E during the induction of membrane curvature. It is quite evident that although a lot of progress has been made in determining the role of E in assembly, much still remains unknown. The role of E has also been proposed to be merely catalytic by functioning to pinch off, or in the scission of, the viral particle from the ER membrane during the terminal phase of budding [63].

Assembly and budding: Scission

The viral envelope is formed primarily during assembly and culminates when the virion buds from the host membrane, a process known as scission [179]. Broadly, enveloped viruses can accomplish membrane scission either by hijacking/exploiting the host cell’s scission machinery or through the expression of their own scission proteins [179]. In the absence of scission machinery, the budding process begins but ultimately stops, and render budding virions attached to the membrane by a small membranous neck. This causes virions to have an uncharacteristically elongated morphology sometimes referred to as “beads-on-a-string” and is seen in viruses that lack the necessary machinery to release the budded virion [179–183]. This is clearly and elegantly demonstrated in the mutation of the matrix-2 (M2) protein, a viral protein responsible for the budding and scission of the influenza virus. Virions that have failed to undergo scission remain attached to the host cell membrane by a membranous neck. The budding process is reinitiated at the site where scission failed, and a new virion is formed. However, the new virion also remains attached to the membrane as well as the previous virion by a small membranous neck. The continuation of this cycle and repeated initiation of budding results in the formation of consecutive scission-defective virions that resemble beads on a string [180, 181]. The same morphology has been reported for the Moloney murine leukaemia virus upon deletion and mutation of p12 protein that functions in its assembly and release [182].

While some enveloped viruses, like influenza A virus, encode their own scission proteins, other viruses rely on the host cell’s endosomal sorting complex required for transport (ESCRT) to accomplish this [179]. This demonstrates a necessity for viral-host PPIs but given the shortage of information available on CoV E-host PPIs, it is nearly impossible to say whether E mediates scission in an ESCRT-dependent manner or not. It is, therefore, essential that host cell candidates capable of interacting with CoV E be identified as they could be potential therapeutic targets for CoV antivirals to stop CoV scission. Conversely, the release of influenza virions is mediated by the M2 protein in an ESCRT-independent manner. The amphipathic helix located in the cytoplasmic tail of the M2 protein is both required and sufficient for the detachment of vesicle buds in an in vitro model system [184]. Mutation of the hydrophobic region of the helix also significantly reduced viral release in vivo, confirming the importance of the 17-amino-acid-helix in the release of the influenza virus in vivo as well. In the absence of the M2 protein, buds formed inside infected cells but failed to detach and such cells exhibited the beads-on-a-string morphology. This suggests that M2 can serve as a substitute for ESCRT complexes during influenza virus budding and, more importantly, raises the possibility of functionally equivalent M2’s in other enveloped viruses.

Interestingly, an amphipathic α-helix is predicted to be located in the TMD of CoV E and has even been confirmed in some of the CoVs [72, 76, 77, 135, 136, 138, 140, 159, 185, 186]. It appears that no attempts have been made to determine whether E of any of the CoVs is responsible for the scission of CoV virions during budding. However, expression of E alone has been reported to produce and secrete vesicles from cells but no further research has been done to determine how this is possible [60, 143]. Mutational studies would certainly benefit from EM analysis to determine what effects TMD mutations of E would have on virion budding. Electron microscopy can clearly demonstrate the consequences of mutated scission proteins and can even prove useful to ascertain what effects complete gene deletion have on viral budding.

Release: Viroporin

While the accumulation of E at the ERGIC points largely to a role in assembly and budding, only a small portion is incorporated into the viral envelope, suggesting that E has additional functions centred around the ER and Golgi region [66, 92, 109, 159]. Viroporins are viral-encoded membrane pore-forming proteins that can modulate cellular ion channels and have been suggested to regulate and function in multiple stages of the viral life cycle, from viral entry to assembly and release, and even pathogenesis [184, 187–196]. Although viroporins are not essential to viral replication, their absence does weaken or attenuate the virus and diminishes its pathogenic effects [35, 197–200]. They tend to be small proteins (~ 60–120 amino acids) of a predominantly hydrophobic nature that oligomerise in the membranes of infected cells, forming hydrophilic pores. The hydrophobic residues line the outside of the structure, oriented toward the phospholipids, while the inside of the pore is made up of the hydrophilic resides [140, 159, 201–204]. Most viroporins share certain structural features such as an amphipathic α-helix in the hydrophobic domain (HD) along with a cluster of positively charged, basic amino acids (such as lysine or arginine) which anchor the pore to the membrane through electrostatic interactions with the negatively charged phospholipids (Fig. 5) [187, 205–207].

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Fig. 5

Illustration of a typical viroporin structure and motifs. The pore of the viroporin (brown) is created by the amphipathic α-helix and the viroporin is anchored to a lipid bilayer by terminal positively charged residues (lysine or arginine). Conformational changes in the structure regulate the flow ions through the viroporin by opening (left) and closing (right) the pore [208]

Viroporins can transport different ions but appear to be largely selective for the positively charged ions hydrogen (H⁺), K⁺, Na⁺, and calcium (Ca²⁺) [209, 210]. Although preferentially selective for cations, viroporins can also transport anions. The preference simply appears to be for cations over anions [211–213]. It is, however, interesting to note that, at a neutral pH, the ion selectivity of the respiratory syncytial virus (RSV) small hydrophobic (SH) protein can change from cationic to anionic [214]. This suggests that viroporins are sensitive to changes in the cellular environment, a property that could be of therapeutic value. After all, the influenza A virus M2 protein is pH-gated and activates upon acidification of the endosome following receptor-mediated endocytosis of the virus [215]. In the same study, Schnell and Chou [215] showed that the anti-viral drug rimantadine exerts its anti-viral property by stabilising the M2 viroporin in its closed conformation and in doing so inhibits viral replication [209, 216]. Similarly, the E protein of several CoVs possesses ion channel activity, though the only structural data of the CoV viroporin has been derived from SARS-CoV using synthetic peptides [75, 135, 136, 138, 217, 218].

Synthetic peptides of SARS-CoV E demonstrate that the TMD is responsible for its ion-conductive properties [135, 136, 138]. Computational predictions and spectroscopic studies show that the SARS-CoV E TMD undergoes oligomerisation, characteristic of ion-channelling proteins, to form a stable pentamer [75, 135–137]. Viroporin formation appears to be mediated by ionic interactions rather than disulphide bonds as mutation of the porcine reproductive and respiratory syndrome virus (PRRSV) E protein cysteine residues appears to be dispensable for oligomerisation [219]. Research into the mechanism of viroporin formation is hampered by the hydrophobic nature of the TMD and has thus far been limited largely to mutational studies and the use of ion channel inhibitors such as amantadine and hexamethylene amiloride.

The CoV E viroporin is equally cation-selective when it comes to its ion-channelling properties, demonstrating a preference for the monovalent cations Na⁺ and K⁺ [217, 218]. Synthetic peptides of SARS-CoV E, that resemble the CoV E viroporin, are able to transport Na⁺, K⁺, and chloride ions (Cl⁻) but are more selective of Na⁺ over K⁺ and least selective of Cl⁻ [217]. Synthetic peptides that correspond to E from HCoV-229E, MHV, and IBV exhibit a similar cation-selectivity for MHV and IBV E as for SARS-CoV E. However, it is interesting that although the E viroporin synthetic peptides of HCoV-229E were still cation-selective, it exhibits a slightly higher selectivity for K⁺ than for Na⁺ [218]. The SARS-CoV E synthetic peptide findings were corroborated using a full-length SARS-CoV E protein [76]. More recently, purified full-length MERS-CoV E has also demonstrated limited ion-channelling properties and would benefit from a more comprehensive characterisation to establish whether it has ion-channelling properties similar to that of the other CoVs [140].

It should be cautioned that the charge on the lipid head group of membranes used can modulate the ion-selectivity of the viroporin. Neutral lipids appear to negate the selectivity of the viroporin as the channels formed did not seem to differentiate cations from anions. In contrast, negatively charged lipids were more cation-selective than neutral lipids, being more permeable to cations [76]. This suggests that the lipid head group of the membranes in use should be taken into consideration when interpreting the results as it might skew the results and inaccurate conclusions may be drawn. At times, the ion channels were only marginally more selective of cations, bringing into question the ion-selectivity of the CoV E viroporin for one cation over another. In fact, an ion channel is only considered ion-specific when its permeability is nearly exclusive to one ion while extremely low to others [220]. Synthetic peptides corresponding to the full-length SARS-CoV E viroporin have also recently been shown to be capable of transporting Ca²⁺ and was linked to the inflammatory response often observed in ARDS [221]. This is the only study so far to have shown that the E viroporin of any CoV is capable of Ca²⁺ transport.

Recent efforts have been directed toward understanding how mutant CoV E viruses carrying ion channel-inactivating mutations revert to their original pathogenic state. Mutants of SARS-CoV E carrying mutations N15A and V25F in the TMD restored ion channel activity by incorporating compensatory mutations in both in vitro and in vivo systems [77]. Mutant N15A reverted by incorporating a single mutation that led to an amino acid change at the same position (A15D), creating a more stable mutant. Conversely, mutant V25F reverted to mutants with amino acid substitutions at either the same position (F25D) or positions relatively close to the original mutation (L19A, F20 L, F26 L, L27S, T30I, L37R). Intriguingly, the V25F mutants appeared as early as 2 days after mice were infected where revertant mutant T30I surpassed the growth of the original virus by day two. This suggests that while some of these mutations appear to merely restore the loss of ion channel activity, it is not entirely inconceivable that revertant viruses would acquire gain of function mutations that can render it more virulent [77]. Similar results were recently reported for IBV E TMD residues analogous to N15A and V25F (T16A and A26F) [222]. It is interesting to note that in both cases SARS-CoV E and IBV E followed a similar trend in their reversion: mutations at N15A and T16A both reverted by substitution of a single residue, whereas mutations at V25F and A26F produced revertants by acquisition of multiple residues.

Some viroporins have been implicated in the release of viruses, but it is not yet known whether the release is mediated by the ion channel activity of the proteins [187, 223–226]. An intriguing study recently reported that both IBV infected and IBV E transfected cells exhibited a marked increase in the pH of the Golgi lumen [227]. These findings suggest that the IBV E viroporin could channel H⁺ and possibly mediate viral release by its ion channel activity. However, this increase in pH was found only in cells expressing a monomeric form of IBV E and not the oligomeric form as required for viroporin formation. The authors proposed that the change in pH could be attributed to an interaction between the monomeric form of E and a host protein. Although possible, only a very small number of host proteins have been shown to interact with CoV E. The monomeric and oligomeric forms were produced by transfection of mutated IBV E A26 to F26 (E^A26F) and T16 to A16 (E^T16A), respectively. In an earlier study, the same authors demonstrated that these two forms were present in IBV E-infected cells but that the monomeric form was much less (~ 10%) in infected cells than in transfected cells (~ 50%). The oligomeric form, however, was the dominant form in infected cells [90]. This suggests that other viral proteins might affect or modulate the oligomerisation of IBV E. It is interesting to note that the M2 protein amphipathic helix motif was required for release of influenza A virus (IAV) particles, perhaps indicating that this motif might be required for the processes budding, scission, and for viroporin activity [181]. It might be worth investigating whether ion-channel inhibitors, such as amantadine, or proton pump inhibitors specifically are able to inhibit this increase in Golgi pH. For now, though, it still remains to be seen whether CoV release is mediated by viroporin ion channel activity or through PPIs with host proteins of the secretory pathway.

Pathogenesis: ER stress response/unfolded-protein response (UPR) and apoptosis

The ER can sustain a high load of protein content without being overwhelmed [228]. However, when the ER’s capacity for folding and processing proteins is exceeded, unfolded or misfolded proteins rapidly accumulate in the lumen and the ER stress response, or unfolded-protein response (UPR), is activated. The various signalling pathways that make up the UPR collectively function by enhancing the folding of proteins, chaperoning, and ER-assisted degradation (ERAD) [229]. If, however, the UPR is prolonged and irreversible, apoptosis will be initiated [230]. By increasing the protein content, folding, and processing of the ER, viral infections can also trigger the UPR and this pathway can be used by the host cell as an antiviral response [231]. Very few studies have looked at the role of CoV E in the ER stress response and its ability to induce apoptosis. In cultured cell lines, overexpressed MHV E and epitope-tagged SARS-CoV E induces apoptosis [87, 232]. However, cells infected with rSARS-CoV and rSARS-CoVΔE, a more biologically relevant system, demonstrated that SARS-CoV E may regulate the UPR as part of its pathogenesis [233]. Cells infected with SARS-CoVΔE exhibit a stronger stress response compared to cells infected with the wild-type virus. Moreover, a higher degree of apoptosis was observed in SARS-CoVΔE-infected cells than in those infected with the wild-type virus.

This study demonstrates the risk of interpreting data from overexpression and epitope-tagged studies. Results generated by such studies might offer some insight into the putative functions of viral proteins but should be interpreted with great care as they can be misleading. Findings can only be more conclusive when supported by results from studies in more biologically relevant systems. The study also shows that CoV E has an anti-apoptotic function in infected cells by suppressing the UPR during infection, likely as a survival mechanism and to continue viral propagation. This function of E has only been demonstrated in SARS-CoV so far, one of the most virulent HCoVs. It would be interesting to see whether E of the other CoVs, as well as the less virulent HCoVs, are also able to contribute to pathogenesis by regulating the host cell stress response.

Funding

No specific funding was obtained for the preparation of the manuscript.