Recombinant Severe acute respiratory syndrome coronavirus Envelope small membrane protein (E)

What is the basic structure and location of the SARS-CoV-2 E protein?

The SARS-CoV-2 E protein is a 75-residue integral membrane protein consisting of a long transmembrane helix (residues 8-43) and a short cytoplasmic helix (residues 53-60) connected by a complex linker that exhibits some internal mobility . NMR spectroscopy studies have revealed that while residues Val14-Thr35 are almost entirely buried in the hydrophobic region of the membrane, Asn15 lines a water-filled pocket that potentially serves as a drug-binding site .

The E protein is primarily located in the viral membrane coat, but most of it is found in intracellular transport sites of the host, including the endoplasmic reticulum (ER), Golgi apparatus, and ER-Golgi intermediate compartment (ERGIC), which are involved in viral assembly and budding processes . Its N-terminus is exposed on the external surface of the virus, which has implications for its interactions with host factors .

How does the E protein sequence of SARS-CoV-2 compare with other human coronaviruses?

Comparative sequence analysis of the E protein across seven known human coronaviruses reveals limited large homologous/identical regions. Only the initial methionine, Leu39, Cys40, and Pro54 are generally conserved across these viruses . SARS-CoV-2 E protein shows the highest sequence similarity with SARS-CoV-1 (94.74%), followed by MERS-CoV (36.00%) .

Interestingly, coronaviruses that typically cause severe disease (SARS-CoV-1, SARS-CoV-2, and MERS-CoV) show significantly higher sequence similarity among their E proteins compared to coronaviruses that cause mild to moderate upper respiratory symptoms typical of the common cold . This observation highlights the potential importance of the E protein in disease severity and development.

What are the established methods for preparing recombinant SARS-CoV-2 E protein for structural studies?

The preparation of full-length SARS-CoV-2 E protein for structural studies, particularly NMR spectroscopy, requires specialized approaches due to its hydrophobic nature. One successful method involves using a ketosteroid isomerase (KSI) fusion partner to facilitate high-level expression of the protein as inclusion bodies in bacterial systems .

A novel purification scheme uses n-hexadecylphosphocholine (HPC; fos-choline-16) micelles to solubilize the protein-containing inclusion bodies. The critical advantage of this method is that the polypeptides are never exposed to any other detergent, lipid, or organic solvent, which would require exchanges or refolding procedures . The complete protocol involves:

• Expression with KSI fusion partner in bacterial systems

• Initial solubilization of inclusion bodies with HPC

• Maintaining HPC throughout all purification steps

• Direct preparation of magnetically aligned bilayer samples suitable for oriented sample solid-state NMR

This approach yields well-resolved solution NMR spectra and overcomes difficulties encountered in previous studies, which were limited to N- and C-terminal truncated constructs missing seven N-terminal residues that are essential components of the drug-binding site .

What is the oligomeric state of the SARS-CoV-2 E protein in membrane environments?

In this dimeric structure, the two helices have a tilt angle of only 6°, resulting in an extended interface dominated by Leu and Val sidechains . The discrepancy between these findings suggests that the E protein may adopt different oligomeric states depending on experimental conditions, membrane composition, or to perform multiple functions during viral infection and replication.

How does the E protein contribute to SARS-CoV-2 pathogenesis and inflammatory response?

The E protein plays multiple roles in coronavirus pathogenesis, particularly in triggering inflammatory responses. Studies have demonstrated that the E protein is capable of inducing pyroptosis-like cell death, rather than classical apoptosis as previously thought . When cells expressing the SARS-CoV-2 E protein were observed, they exhibited swelling followed by explosion and release of cellular contents, characteristic of pyroptosis .

The E protein has two conserved key features that contribute to pathogenesis:

• An N-terminal region with putative ion channel activity

• A C-terminal PDZ-binding motif

These features play key roles in inducing an inflammasome response leading to acute respiratory distress syndrome (ARDS), a major cause of death from coronavirus infections . The ion channel activity may be further modulated by the charge of the lipid membranes, providing additional flexibility for viral replication under various conditions .

Additionally, the E protein has been found to interact physically with Toll-like receptor 2 (TLR2) in a specific and dose-dependent manner, engaging the TLR2 pathway to activate NF-κB transcription factor and stimulate the production of CXCL8 inflammatory chemokine . This interaction may contribute to the cytokine storm observed in severe COVID-19 cases.

What experimental evidence exists for the role of E protein in viral assembly and replication?

Multiple lines of experimental evidence demonstrate the critical importance of the E protein in viral assembly and replication:

• Virion morphology studies: Recombinant coronaviruses lacking the E gene (ΔE) display abnormal morphology, with vesicles containing dense, granular material interspersed between virions, believed to be immature virions resulting from an aborted viral budding process .

• Viral titer reduction: Deletion of E proteins from SARS-CoV-1 (rSARS-CoV-1-ΔE) resulted in a 20- to 200-fold reduction in virus production . Similarly, in vitro studies have shown that removal of the E protein from recombinant coronavirus particles attenuates viral maturation and produces incompetent progeny .

• Structural alterations: When the C-terminal residue of mouse hepatitis virus (MHV) E protein was mutated to alanine, the virion adopted a contracted, elongated shape rather than the typical spherical particles observed in wild-type virions .

• Membrane interactions: The E protein is critical for the formation of viral protein-containing vesicles in SARS-CoV-1 and SARS-CoV-2, occurring mainly in pentameric form in these contexts . This activity appears essential for proper viral budding and membrane curvature.

These findings collectively establish that while coronaviruses can form without E protein, the resulting particles are significantly less infectious and improperly assembled, confirming E protein's crucial role in viral morphogenesis and maturation.

How do SARS-CoV-2 E protein interactions with host proteins impact cellular function?

The SARS-CoV-2 E protein interacts with several host proteins, significantly affecting cellular functions and contributing to viral pathogenesis:

• TLR2 Interaction: E protein binds directly and physically with TLR2 in a dose-dependent manner . This interaction activates the NF-κB transcription factor and stimulates CXCL8 production. Chemical inhibition of NF-κB led to significant inhibition of CXCL8 production, while blockade of P38 and ERK1/2 MAP kinases resulted only in partial CXCL8 inhibition . This interaction likely contributes to the inflammatory response during infection.

• PALS1 Interaction: Molecular docking studies have shown that SARS-CoV-2 E protein binds to PALS1 with higher affinity than SARS-CoV-1 E protein . The deletion of glutamic acid 69 (E69) and glycine 70 (G70) residues from SARS-CoV-1 E and their substitution with arginine 69 (R69) in SARS-CoV-2 E enhances binding between SARS-CoV-2 E and PALS1 . The Gibbs free energy calculations demonstrate that SARS-CoV-2 E (-97.10 kcal/mol) has a higher affinity for PALS1 than SARS-CoV-1 E (-63.62 kcal/mol) . This stronger interaction could disrupt the pulmonary epithelial barrier more effectively, amplifying the inflammatory process.

• ER Stress Response: E protein can trigger the ER stress response in host cells, which may contribute to cellular dysfunction and death .

These interactions demonstrate how the E protein functions not only as a structural component but also as a virulence factor that manipulates host cellular processes to enhance viral replication and pathogenesis.

What potential therapeutic strategies target the SARS-CoV-2 E protein?

Several therapeutic strategies targeting the E protein have been proposed based on its crucial role in viral replication and pathogenesis:

• Ion Channel Inhibitors: Amilorides have been identified as potential inhibitors of E protein ion channel activity . NMR studies have characterized the interactions of amilorides with specific E protein residues and correlated this with their antiviral activity during viral replication. The binding affinity of amilorides to E protein correlated with their antiviral potency, suggesting that E protein is indeed the likely target of their antiviral activity .

• Targeting the Drug-Binding Pocket: The water-filled pocket lined by Asn15 identified in structural studies represents a potential drug-binding site that could be exploited for therapeutic development .

• Neutralizing Antibodies: Given the E protein's role in pathogenesis, exploring the therapeutic effect of anti-E blocking or neutralizing antibodies in symptomatic COVID-19 patients has been proposed .

• Vaccine Development: The E protein has been suggested as a promising non-Spike SARS-CoV-2 antigen candidate to include in the development of next-generation prophylactic vaccines against COVID-19 infection and disease .

• TLR2 Pathway Inhibition: Given the E protein's interaction with TLR2 and subsequent inflammatory response, inhibitors of this pathway could potentially reduce inflammatory complications of COVID-19 .

These strategies highlight the potential of the E protein as a therapeutic target, particularly for reducing inflammation and inhibiting viral replication.

How do mutations in the E protein affect SARS-CoV-2 function and host interactions?

Mutations in the E protein can significantly affect viral function and host interactions, as demonstrated by comparative studies between SARS-CoV-1 and SARS-CoV-2:

• Enhanced Host Protein Binding: The substitution of glutamic acid 69 (E69) and glycine 70 (G70) in SARS-CoV-1 E with arginine 69 (R69) in SARS-CoV-2 E enhances binding to PALS1 . The acquisition of R69 produces a salt bridge and several hydrogen bonds between E and the PALS1 binding pocket, strengthening this interaction. In contrast, the small sidechain of G70 in SARS-CoV-1 E prohibits the formation of such bonds, reducing the strength of the E-PALS1 interaction .

• Altered Virion Morphology: Mutations in the C-terminal region of the E protein in mouse hepatitis virus (MHV) led to virions with contracted, elongated shapes rather than the typical spherical particles observed in wild-type virions, demonstrating how E protein mutations can affect viral assembly and morphology .

• Oligomeric State Changes: Different mutations may affect the oligomeric state of the E protein, potentially switching between dimeric and pentameric forms, which would impact its function as an ion channel and/or its role in membrane curvature during budding .

• Pathogenicity Modulation: Given the high sequence similarity between E proteins of coronaviruses causing severe disease compared to those causing mild symptoms, specific mutations may contribute to increased pathogenicity and more severe clinical outcomes .

Understanding these mutation effects provides insights into viral evolution and might inform therapeutic strategies targeting conserved regions less prone to functionally significant mutations.