The membrane topology of the E protein has been studied in a number of coronaviruses with inconsistent results; the protein's orientation in the membrane may be variable.[3] The balance of evidence suggests the most common orientation has the C-terminus oriented toward the cytoplasm.[8] Studies of SARS-CoV-2 E protein are consistent with this orientation.[5][9]
In some, but not all, coronaviruses, the E protein is post-translationally modifiedbypalmitoylation on conserved cysteine residues.[2][8] In the SARS-CoV E protein, one glycosylation site has been observed, which may influence membrane topology;[8] however, the functional significance of E glycosylation is unclear.[2]Ubiquitination of SARS-CoV E has also been described, though its functional significance is also not known.[2]
Studies in different coronaviruses have reached different conclusions about whether E is essential to viral replication. In some coronaviruses, including MERS-CoV, E has been reported to be essential.[10] In others, including mouse coronavirus[11] and SARS-CoV, E is not essential, though its absence reduces viral titer,[12] in some cases by introducing propagation defects or causing abnormal capsid morphology.[2]
Virions and viral assembly
[edit]Illustration of a coronavirus virion in the respiratory mucosa, showing the positions of the four structural proteins and components of the extracellular environment[13]
The E protein is found in assembled virions where it forms protein-protein interactions with the coronavirus membrane protein (M), the most abundant of the four structural proteins contained in the viral capsid.[2][4] The interaction between E and M occurs through their respective C-termini on the cytoplasmic side of the membrane.[2] In most coronaviruses, E and M are sufficient to form virus-like particles,[2][4] though SARS-CoV has been reported to depend on N as well.[14] There is good evidence that E is involved in inducing membrane curvature to create the typical spherical coronavirus virion.[2][15] It is likely that E is involved in viral budding or scission, although its role in this process has not been well characterized.[2][4][15]
Viroporin
[edit]The E viroporin opens at acid pH. The open state in pink presents a wide N-terminus. Conversely, the C-terminus narrows in the open state, which brings the polar sidechains of Thr35 and Arg38 close to the hydrophobic gate at Leu28. This presumedly lowers the energy barrier for ions to cross the channel.
In its pentameric state, E forms cation-selective ion channels and likely functions as a viroporin.[5] NMR studies show that viroporin presents an open conformation at low pH or in the presence of calcium ions, while the closed conformation is favored at basic pH.[16] The NMR structure shows a hydrophobic gate at leucine 28 in the middle of the pore. The passage of ions through the gate is thought to be facilitated by the polar residues at the C-terminus.[17]
The E protein's role as a viroporin appears to be involved in pathogenesis and may be related to activation of the inflammasome.[3][18] In SARS-CoV, mutations that disrupt E's ion channel function result in attenuated pathogenesis in animal models despite little effect on viral growth.[10]
The sequence of the E protein is not well conserved across coronavirus genera, with sequence identities reaching under 30%.[12] In laboratory experiments on mouse hepatitis virus, substitution of E proteins from different coronaviruses, even from different groups, could produce viable viruses, suggesting that significant sequence diversity can be tolerated in functional E proteins.[20] The SARS-CoV-2 E protein is very similar to that of SARS-CoV, with three substitutions and one deletion.[4] A study of SARS-CoV-2 sequences suggests that the E protein is evolving relatively slowly compared to other structural proteins.[21] The conserved nature of the envelope protein among SARS-CoV and SARS-CoV-2 variants has led it to be researched as a potential target for universal coronavirus vaccine development.[22][23]