Overview
The envelope (E) protein of Sars-CoV and Sars-CoV-2 is the smallest of the viruse’s structural proteins. It is an integral membrane protein embedded in the envelope, but also localized in the ER, golgi and ERGIC, once a host cell has been infected [1].
The structure of the envelope protein of Sars-CoV has so far only been modeled based on nuclear magnetic resonance (NMR) data. Yet, modeling the 3D structure of Sars-CoV-2 E protein turns out to be quite challenging. Only the amino acid sequence of Sars-CoV-2 E protein is known, yet. Hence, the structure and function of Sars-CoV-2 E protein can only be prdicted by comparison with the Sars-CoV E protein.
Sequence alignements of the envelope proteins of both viruses, consisting of 76 amino acids in Sars-CoV and 75 amino acids in Sars-CoV-2 [2] [3], demonstrate a 91% sequence homology [2]. Functional and structural comparison of Sars-CoV-2 with the existing knowlege about Sars-CoV could provide a good prediction of the E protein’s role in causing Covid-19.
Structure
The E protein’s topology is mainly separated into three domains: A short hydrophilic N-terminus, that is identical in Sars-CoV and Sars-CoV-2
[3] and works as a golgi-targeting signal, a long hydrophobic transmembrane domain (TMD) and a long hydrophilic C-terminus containing an β-coil-β-motif as well as a PDZ-binding motif (PBM) at residues 73-76
[3]. It still remains unclear wether the C- and the N-terminus are luminal or cytoplasmic. Studies on this question exhibit different results depending on the software modeling tool they used. This may lead to the assumption that membrane topology might differ depending on the E protein’s multiple functions or on the level of its expression or oligiomerization [3].
Different structural features are incorporated in the secondary structure of E. The monomeric form of the protein consists of a long amphipathic α-helix forming the TMB domain and a short α-helix in the C-terminus which is believed to be in a dynamic equilibrium with the less abundant β-coil-β-motif [4]. Both helices are connected by a turn structure [5]. The β-coil-β-motif including a conserved proline at residue P54 in Sars-CoV and Sars-CoV-2 is proposed to function as a golgi targeting signal and to switch its conformation to alter the E protein’s function in the host cell [4]. Unfortunately, there are no pdb structures available that display the predicted β-coil-β-motif. Apart from the monomeric form of the protein, E also shows to oligiomerize to shape a pentameric ion channel in the host cell’s membranes. Oligiomerization is induced by the long amphipathic α-helix of the TMD [3].
Posttranslational modifications
Several posttranslational modifications are proven to modify the E protein of Sars-CoV and other viruses. Palmitoylation is the addition of palmitic acid to cystein residues increasing the protein’s hydrophobicity. Hence, palmitoylation of E assists in membrane association and anchoring. In addition, the modification is suggested to co-localize the virus to the golgi membrane by operating as an additional targeting sequence.
Ubiquitination assays with Sars-CoV E protein proves that its “ubiqutination status inversely correlates to its stability and half life”[3]. Accordingly, ubiquitination might function as a negative regulation of E protein levels and consequntly of viral production to achieve the best viral titre.
Another modification shown to occure in Sars-CoV E protein adds oligiosaccharid fragments to asparagine residues in a certain motif (Asn-X-Ser/Thr) which is also involved in the sequence of Sars-CoV-2 envelope protein. This modification, namely glycosilation, recruits chaperone proteins of the host cell that aid in the correct folding of new synthesized viral proteins. In Sars-CoV residue N66 embedded in the motif Asn-Ser-Ser was discovered to be glycosilated, while the other potential glycosilation target N48 also located in a suitable motif (Asn-Val-Ser) had no attached oligiosaccharid. Both motifs are also found in Sars-CoV-2. Experimantal data suggest that glycosilation of N66 might prevent oligiomerization of E to promote various processes induced by monomeric E protein [3].
Function
Viral assembly
Viral assembly means the process of gathering around all the viral molecules (proteins and genome) to form a virus like particle (VLP) resulting in the release of a fully matured virus. The assembly is located at the ERGIC where the VLP buds into the lumen of ERGIC and follows the way through the host cell’s secretory pathway. Several experiments confirm the involvement of the envelope protein in the assembly process. Nevertheless, lack of E in Sars-CoV infected cells still does not stop VLP production completely, but rather cripples viral maturation. In Sars corona viruses lacking the E protein a deviant morphology of virions being pinched and elongated has been observed. The absence of E furthermore arrests virus trafficing and blocks its secretion, thus resulting in a lower number of mature virions, but more vesicles carrying granular material of the aborted assembly. The result is a high rate of propagation-incompetent virions[3].
Membrane curvature
The mechanism behind the E protein’s contribution to viral assembly raises the suspection of E being rather involved in membrane curvature and scission, whereas the structural membrane protein (M) coordinates viral assembly. Yet, it still needs further investigation to analyse the exact mechanism behind membrane formation of virions. A potential mechanism was proposed to resemble the mechanism viral proteins nsp 3 and nsp 4 use to induce ER membrane rearrangement and membrane curvature of double membrane vesicles (DMV). Nsp 3 and 4 feature a secondary structure called “luminal loop” containing a cystein residue that may be palmitoylated in order to anchor to a membrane during membrane curvature. Given that E can exhibit multiple topologies, further research may concentrate on proving, if the protein could also adopt “luminal loops” as a structural feature. An alternative mechanism suggests membrane curvature being induced by homotypic interactions of the E-protein’s N-termini [3].
Membrane Scission
The viral assembly is followed by the VLP finding its way through the secretory pathway and ends in the release of the mature virus from the host cell. The process of detaching from the host membrane is known as scission, which is either coordinated by the viruse’s own scission proteins or by the host cell’s scission machinery (ESCRT). Infected cells lacking the scission machinery exhibit a “beads-on-a-string” morphology, according to the virions beeing stuck to the host membrane and causing an elongated shape. E is proposed to be involved in membrane scission due to its amphipathic helix located in the TMD, which is also found in protein M2 required for the release of vesicle buds in influenza viruses. Due to a low number of research attempts, it still remains unclear wether membrane scission in Sars-CoV and Sars-CoV-2 is ESCRT-dependent or not[3].
Ion channel activity
The envelope protein in its oligiomeric form concentrates hydrophilic residues of the TMD in the inside of the channel structure, whereas the hydrophobic amino acids orientate towards the phospholipids of the membrane [6]. Thus, specific structural features are required for anchoring the viroporin to the membrane, namely an amphipathic α-helix as well as basic positively charged residues. The anchoring process is mediated by electrostatic interactions between the positive amino acids and the negatively charged phospholipids. Pores of Sars-CoV E protein mainly favour the transport of Na+ and K+, but were also found to be permeable for Ca2+. Ion selectivity is suggested to be generated by residue N15 used as a sort of filter [7] and can further be affected by the charge of the membrane’s lipid head group.
Ion channels involving the inactivating mutations N15A and V25F in the TMD display to regain their original virulence by incorporating other single mutations at the same position (A15D, F25D) or multiple changes at several positions nearby (L19A, F20 L, F26 L, L27S, T30I, L37R).
“This suggests that while some of these mutations appear to merely restore the loss of ion channel activity, it is not entirely inconceivable that reverant viruses would acquire gain of function mutations that can render it more virulent”[8]. Deletion of the envelope protein in its pentameric state demonstrates that ion channel activity is not essential for viral replication, but yet attenuates the virulence.
Even though the primary function of transporting sodium and potassium cations is not yet clear, Ca2+ is proposed to trigger the inflammatory response seen in acute respiratory distress syndrome [9]. Additionally, the E protein oligiomer of Infectious Bronchitis Virus (IBV) has already been observed to be involved in virus release. The release may be induced by the measured pH increase in the golgi lumen eventually caused by the viroporine channeling H+. In Influenza A Virus (IAV) infected cells however, VLP release was promoted by a PPI via an amphipatic α-helix motif. For the viral release of Sars-CoV and Sars-CoV-2 both mechanisms are possible models that need experimantal verification [3].
Intracellular trafficing and targeted localization
The structure of the E protein includes a golgi-targeting signal in the β-coil-β motif of the C-terminus and another one in the N-terminus. Additionally, palmitoylation as a posttranslational modification is believed to be involved in the golgi targeting process as well. The targeting signal locates the E protein to the golgi membrane, where E monomers can oligiomerize and form a viroporine in the golgi membrane. From there, the virus acquires the membrane for a new envelope and finds its way through the secretory pathway of the host cell[3].
Pathogenesis
As a part of the host cell’s viral defense, the ER stress response is activated, once the protein folding capacity of the ER is overloaded by additionally expression of viral proteins. The ER stress response, also called unfolded protein response (UPR), can lead to apoptosis of the host cell. Sars-CoV infected cells lacking the envelope protein display to have a strong stress response, whereas the wild-type virus exhibts a milder UPR and a lower level of apoptosis. This may give rise to the assumption that the E protein contributes to pathogenesis by suppressing the ER stress response and apoptosis to maintain the survival of the host cell[3].
Interference of viral proteins with the immune system is often observed and also seen in Sars-CoV infected cells. Thereby the ion channel activity of E is a crucial factor that activates the inflammatory pathway by channeling Ca2+ [9] resulting in lung damage in infected mice. Inhibition of the viroporine by hexamethylene amiloride (HMA) [7] reduces the activation of the inflammasome, what makes the ion channel of E a potential therapeutic target.
Protein-Protein-Interactions (PPI)
PPIs of the viruse’s proteins among each other and interactions with host cell proteins occur to deregulate a numerous amount of physiological processes. This causes pathogenesis with several symptoms observed in patients suffering a Sars-CoV infection[3].
Interactions of E with viral proteins
The small envelope protein unveils to interact with other structural proteins expressed in Sars-CoV as well as with the E protein itself. To form a pentameric viroporin functioning as an ion channel, E monomers interact with their long α-helix in the TMD and oligiomerize. The PPI is mainly mediated by residue V25 and additionally by residue N15 being slightly involved. Both residues are present in Sars-CoV-2 aswell. Interacting with the SARS-CoV-2 protein M via the C-termini of both at the cytoplasmic side of ERGIC, E aids in VLP formation and release, whereas deletion of both interacting domains reduces VLP formation. The SARS-CoV-2 protein N seems to interact with the envelope protein independently from M. Nevertheless, co-expression of N with envelope and membrane protein further enhances the VLP production while the exact mechanism and reason of this PPI has not yet been revealed. Both C-termini of E and of N induce this interplay. However, interaction with SARS-CoV-2 protein S is suggested to be mediated by disulfide bonds between cystein motifs appearing in E and S of Sars-CoV and Sars-CoV-2. A study using tandem affinity purification and mass spectrometry has proved that S is being co-purified with E, even though they did not propose a possible mechanism or function of this PPI [10]. The last proven viral-intern interaction with E is provided by the structural protein 7a, known to be specifically expressed in Sars-CoV. Protein 7a is present in mature virions and arrests the cell cycle, induces apoptosis and triggers the expression of pro-inflammatory cytokines in the host cell. Yet, the reason of E interacting with 7a and the presence of Protein 7a in Sars-CoV-2 remains unknown[3].
Interactions of E with host proteins
Interactions of the envelope protein with proteins of the host cell are mediated by its PBM domain at the very end of the C-terminus. Among all corona viruses the PBM domain slightly differs, but is identical in the E proteins of Sars-CoV, BatCoV and Sars-CoV-2[2] containing the four residues DLLV. The motif binds to the PDZ domain of adaptor proteins subsequently bound by other cellular proteins. Followingly, a signalling cascade that possibly causes pathogenesis is activated. Five interactions between E and host proteins are reported until now. The anti-apoptotic B-cell lymphoma-extra-large (Bcl-xL) protein is proposed to cause SARS-CoV-induced lymphopenia [11]. Another interacting partner of the envelope protein has been identified as protein associated with C. elegans lin-7 protein 1 (PALS1) which was found to disrupt tight junctions of pulmonary epithelia cells in lungs. This eventually consults in an epithelial barrier breakdown and virions breaking through the alveolar wall causing a systemic infection [12]. ”The breakdown of the epithelial barrier is a hallmark in respiratory distress syndromes” [13]. Interaction of syntenin with E caused its transportation to the cytoplasm where the protein triggered the overexpression of inflammatory cytokines. A proposed consequence may be an overreaction of immune response effectuating tissue damage, oedema and acute respitory distress syndrome (ARDS) [14]. Both, (Na+/K+) ATPase α-1 subunit and stomatin are cellular proteins that contribute to sustain ionic homeostasis. Interacting with the viruse’s envelope protein may decrease levels and activity of human epithelial sodium channels required for Na+ transport. Changes in the equilibrium affect fluid volume, blood pressure and water homeostasis [3].
Still, according to predictions there are a lot more interacting partners of E that have not yet been uncovered. As it has already been demonstrated by the known interacting partners of E, PPIs play a crucial role in the pathogenesis. Consequently, further research is necessary to perceive more details about the triggered signalling pathways and to discover new interacting proteins.
See also
Coronavirus_Disease 2019 (COVID-19)
SARS-CoV-2_virus_proteins
COVID-19 AlphaFold2 Models
