Advances in Understanding the Envelope Protein in Coronavirus Infection
Aakanksha Agarwal1 and Ashley L. Steed1*
1Department of Pediatrics, Washington University School of Medicine, Saint Louis, Missouri, United States of America
Abstract
The COVID-19 pandemic continues to impart devastating effects on human health, healthcare systems, and the economy. Vaccination, monoclonal antibodies, and antiviral therapies prevent and limit early infection. Unfortunately, few strategies exist to mitigate the disease burden in the vast number of individuals who seek medical attention with established infection and severe disease. While we have a limited understanding of the mechanistic basis by which SARS-CoV-2 causes critical illness, increasing evidence suggests that host-pathogen interactions shape immune responses that drive the pathogenesis of COVID-19. Therefore, it is imperative to understand the roles of the viral proteins and how they shape the course of infection. One interesting protein is the envelope (E) protein of SARS-CoV-2; this tiny structural protein has been implicated in many phases of the viral life cycle. Importantly, the E protein facilitates viral packaging and replication, and its deletion reduces viral pathogenicity. The E protein also possesses ion channel functions, interacts with host proteins, and has the potential to have various structural topologies. This review aims to establish an updated understanding by highlighting recent developments in the investigation of the SARS-CoV-2 E protein, particularly in comparison to the envelope protein of SARS-CoV. thorough knowledge of this protein will enable targeted studies in hopes of tailored efficacious treatments.
Introduction
Coronaviruses (CoVs) are a large family (Coronaviridae) of enveloped, positive-sense single-stranded RNA viruses that range in size from 28 to 32 kilobases and infect mammals and birds. Seven members of this family are known to infect humans, who develop symptoms ranging from those of a common cold to bronchiolitis to fatal pneumonia with acute respiratory distress syndrome1-5. In December 2019, a novel coronavirus, known as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was discovered in patients with pneumonia in Wuhan, China6-7. SARS-CoV-2 swiftly spread and immediately emerged as a threat to public health on a global scale. In contrast to earlier coronavirus outbreaks of SARS-CoV and , this new coronavirus rapidly swept over the globe and profoundly impacted health, healthcare delivery systems, and the economy. As a result, the World Health Organization classified coronavirus disease 2019 (COVID-19) as a pandemic in March 20208.
SARS-CoV-2 contains four structural proteins, spike (S), nucleocapsid (N), membrane (M), and envelope (E), all of which ensure the formation of mature virions9-10. The most crucial protein for viral entry into cells is the S protein, which attaches to the host surface receptor and has immune recognition sites11-12. Different CoVs employ distinct host entrance strategies utilizing the S protein, pointing to how variations in the S protein's amino acid residues control cell entry. This characteristic is capitalized on during vaccine development13. The N protein wraps the viral RNA into the helical ribonucleocapsid. Its importance is highlighted by the necessity of N in the viral replication cycle and the host cell’s response14. Additionally, it has been demonstrated that the N protein plays a significant role in viral envelope conformation in some CoVs as well as increases the generation of virus-like particles (VLPs)15. The M protein is the most abundant structural protein and gives the viral envelope its form. This 222 amino acids protein has three transmembrane domains with three N- and C-termini accessible within and outside the viral particle. Stable binding of M to the other structural proteins causes the inner core of SARS-CoV-2 to develop, facilitating viral assembly16. The SARS-CoV-2 E protein is a single membrane-spanning protein that contains 75 amino acids with an uneven distribution of charged residuals on both sides of the membrane17-18. The E protein of SARS-CoV-2 plays a crucial role in the virus life cycle and disease pathogenesis. It can form an ion channel or viroporin, leading to host death and cytokine storms19. Therefore, it serves as a promising therapeutic target.SARS-CoV-2 E gene, structure, localization, role in pathogenesis, interactions with the host, and role of therapeutic strategies in light of mutant strain development.
SARS-CoV-2 genome
In comparing the SARS-CoV-2 genome to its most closely related human coronavirus, nearly of the SARS-CoV is identical and more than 90% of the critical enzymes and structural proteins are conserved. The first two-thirds of the coronavirus genome located at the 5′ end, consists of ORFs 1a and 1b which encode two polyproteins that make sixteen non-structural proteins (nsps). The last third of the genome located at the 3′ end encodes the four structural proteins: S, E, M, and N proteins, all of which are necessary to generate a structurally complete viral particle. The genome also contains accessory genes that are located throughout the genome21. (Figure 1)
Figure 1: Schematic representation of (a) human coronavirus particle and (b) SARS-CoV-2 genome.
Envelope protein sequence and structure
SARS-CoV-2 E protein is the smallest of the structural proteins and its overall sequence is similar to E (94.7%), followed by MERS CoV (36%)22. However, a comparison of the E protein sequences of SARS-CoV-2 and the other known human coronaviruses revealed no major homologous sections with only the beginning methionine, Leu39, Cys40, and Pro54 being typically conserved23. It is interesting to note that coronaviruses that cause more severe illness have a higher E genetic sequence similarity to each other than those that cause mild to moderate upper respiratory symptoms.
The three primary domains -CoV E protein structure comprise the following: a short hydrophilic luminal-oriented N-terminal domain (NTD) followed by a long α-helical hydrophobic transmembrane domain (TMD), and a long hydrophilic cytoplasmic C-terminal domain (CTD)24-25. (Figure 2a) The C-terminal region of SARS-CoV E is predominantly an α-helical and contains a PDZ-binding motif (PBM), which has been recognized as a major virulence factor26-27. The PBM comprises the final four carboxy-terminal amino acids (DLLV) and is the most well-studied contact site. The proposed homo-pentameric structure of the E protein is shown in Figure 2b through the membrane (top view) and with the membrane (side view). Interestingly, the SARS-CoV E protein has also been described in a variety of topologies; nonetheless, it lacks a typical cleavage signal sequence and is thus most likely a type II or type III membrane protein28.
A study by Kuzmin et al. found that both monomeric and pentameric E can cause membrane curvature via amphiphilic C-terminal region. This function is responsible for forming a potential budding site on the convex part of the membrane, which helps in the facilitation of virion budding29. it is interesting to consider that different topologies of CoV E protein may impart distinct functions throughout the viral replication cycle30. This idea is worthy of further examination as it would impact therapeutic development.
Figure 2: Characteristics of SARS-CoV E protein. (a) Amino acid sequence alignment of the E protein of SARS-CoV-2 and SARS-CoV. Change of four amino acids are shown in red boxes. (b) Proposed structure of SARS-CoVs homo-pentameric E protein, an ion channel viroporin.
E protein in viral pathogenesis
Protein E is widely expressed inside the infected cell and is essential to produce viral progeny31, although not represented in the mature virions at levels comparable to S or M proteins32. Recombinant coronaviruses deficient in the E gene exhibit much lower viral titers, less viral maturation spread in vitro33. It is likely that protein E functions as a crucial virulence element because SARS-CoV is likewise attenuated in vivo, showing decreased pathogenicity34 and an inability to proliferate in the central nervous system. The pathogenicity of the SARS-CoV-2 virus is also greatly decreased by dominant-negative mutations35. A study by Park et al. demonstrated that hexamethylene amiloride and ethyl isopropyl amiloride have antiviral activity against SARS-CoV-2 by binding to the N-terminal region of the E protein36. Therefore, further study on protein E is paramount.
E protein localization
Coronaviruses differ from many other enveloped viruses (which are often assembled at the plasma membrane) in that they assemble in the ER-Golgi intermediate compartment (ERGIC)37. Infectious virions proceed via the host secretory route into the lumen of the ERGIC after which they are eventually released from the infected cell38. Notably, the bulk of the E protein is found in sites of intracellular trafficking (the ER, Golgi, and ERGIC), where it contributes to CoV assembling, intracellular trafficking, and budding24,28,39.
To understand how SARS-CoV-2 enables the formation of new infectious viral progeny, it is crucial to identify the region of the E protein necessary for targeting the ERGIC. Prior work in SARS-CoV implicates that the E protein's C-terminus contains routing information to the Golgi complex. Additional investigation queried the potential of Golgi-targeting information in the E protein’s N-terminus using an N-terminus chimeric protein40. Interestingly, the N-terminus chimaera was also directed to the Golgi area, suggesting that both the N- and C-termini regions may have targeting information. Although it has been suggested that epitope-tagged E proteins may alter its localization, accumulation in the ER-Golgi in both cells transfected with a tagged E and those infected with virus indicate that the tag had no bearing on E’s localization24,41. Studies examining the localization of E have solely employed FLAG-tagged forms of the protein; however, the inclusion of bulker, such as glutathione S-transferase and green fluorescent protein, may interfere with the localization of the CoV E protein.
Functions and protein-protein interactions
The E protein of SARS-CoV-2 has several functions that help the virus replicate and therefore is considered a potential therapeutic target. While initial work has implicated that the SARS-CoV-2 E protein has a role in virion assembly and release, further work has shown a role in activating the inflammasome as a viroporin22. Thus far investigations have primarily focused on interactions with both viral and host proteins, implicating its role in disease pathogenesis31. Indeed much of its hypothesized functions are extrapolated from work on SARS-CoV. Though the interaction between E and M has been documented, recent investigations have discovered new protein interactions with the SARS-CoV E protein37. In one investigation, cells were infected with SARS-CoV that had a C-terminally tagged E. Tandem affinity purification and tandem mass spectrometry were employed to identify interacting proteins. This study found that the N-terminal ubiquitin-like domain-1 of SARS-CoV nsp3 mediates contact with SARS-CoV E. During infection, nsp3 and SARS-CoV E colocalize, and nsp3 may be responsible for SARS-CoV E ubiquitination42. Another study identified that the C-terminal domain of SARS-CoV E, which encodes the PDZ domain, can recognize human cell junction protein PALS143-46, tight junction protein zonula occludens-1 (ZO-1)47, adhesion junction protein syntenin48, and other connexins. PALS1, ZO-1, and syntenin, have been shown to interact with the E protein. When comparing the E proteins from SARS-CoV and SARS-CoV-2 for affinity to PALS1 using equilibrium and kinetic binding tests, an enhanced affinity for the SARS-CoV-2 E protein was discovered46. E protein’s interactions with ZO-1 and syntenin also cell polarity, which may lead to leaky pulmonary epithelial barriers, increase local viral entry of inflammatory cells into lung alveolar spaces. These findings have led to the hypothesis that E protein’s interactions with host proteins may increase disease pathogenesis. Previous studies have shown that the E protein PBM motif of both SARS-CoV and SARS-CoV-2 can mediate alterations in the host gene expression of proteins related to ion transport and cell homeostasis. a decrease in the mRNA expression of the cystic fibrosis transmembrane conductance regulator (CFTR), essential for edema resolution, has been observed. Researchers have also found that small molecule CFTR modulators can reduce the replication of SARS-CoV-2 in cultured cells and protect against severe disease in a mouse model. These findings highlight the importance of the E protein PBMs in CoV replication and virulence and suggest new cellular targets for the development of antiviral drugs49.
Protein E has also been implicated of the immune response. The hyperinflammatory response in acute respiratory distress syndrome, a potentially deadly consequence, is linked to the production of the E protein. High levels of the inflammatory cytokines interleukin (IL)-1β and IL-6, which are partially mediated by the SARS-CoV E protein, generate a cytokine storm that is considered crucial to this immunopathology50. SARS-CoV E protein induces a more robust inflammatory response compared to SARS-CoV-2 E protein. A recent study revealed that and MYD88 levels are linked to COVID-19 severity, triggering inflammatory responses even in the absence of viral entry. TLR2 senses the SARS-CoV-2 envelope protein as its ligand and blocking downstream signaling can protect against the pathogenesis of SARS-CoV-2 infection. The interaction between the E protein and TLR2 triggers the activation of the transcription factor NF-κB, which in turn stimulates the production of CXCL8 chemokine. These findings suggest that the SARS-CoV-2 E protein may play a role in the increased levels of pro-inflammatory cytokines associated with the development of COVID-1951,52. A recent study has found that the , a vital host factor for SARS-CoV-2 entry, is capable of directly binding to the recombinant E and M proteins, but not S or N proteins. E protein serves as a primary pathogen-associated molecular pattern for TLR1, significantly activating inflammatory responses in myeloid cells53. Moreover, the SARS-CoV-2 E can trigger a strong immunological response in vitro and in vivo that is dependent on type I interferon signaling54.
The cytokine dysregulation has been partly attributed to the interaction of the SARS-CoV E protein with syntenin as well as its viroporin activity50. SARS-CoV E can act as a viroporin with ion channel and membrane-permeabilizing abilities55. Specifically, the ion-channel activity of the SARS-CoV E protein allows it to transport several ions, including Naâº, Kâº, Cl¯, and Ca2âº56. As cell survival depends on maintaining ion homeostasis, these ion channels created by the E protein may harmfully disrupt the electrochemical gradient of host cells. It also creates a cation-selective pentameric ion channel in the ERGIC membrane, which may also promote cell death and hyperinflammatory responses55,57. The role of E as a viroporin and its significance to COVID-19 immunopathology was substantiated by a reduction in IL-1β, IL-6, and TNF-α production when the E protein’s ion channel activity was inhibited52. Notably, electrolyte abnormalities, such as lower blood potassium, sodium, and calcium concentrations, are linked to disease severity in COVID-19 patients58.
Mutations in SARS-CoV-2 E
A global investigation of SARS-CoV-2 genotypes revealed numerous mutations in different parts of the virus such as structural, non-structural proteins, auxiliary proteins, and untranslated regions59. A recent study revealed that SARS-CoV-2 has gone through 8309 single mutations and introduced the mutation ratio and mutation h-index to describe the protein conservation. They found that the SARS-CoV-2 E protein, main protease, and endoribonuclease protein are relatively conserved, whereas the N protein, S protein, and papain-like protease are relatively.After analyzing 15,140 SARS-CoV-2 genomes, only 52 mutations were detected in the E protein gene, indicating an extraordinary low mutation rate60. Within its 225 nucleotides, the CTD of the SARS-CoV-2 E gene has the highest probability of missense mutations, followed by the TMD61. Moreover, mutations in the TMD of the E protein can render it non-functional62. As a relatively stable and conserved protein, further work is necessary to maintain genomic surveillance while also capitalizing on its usefulness as a therapeutic target.
Concluding remarks
Understanding the SARS-CoV-2 virus and its pathogenesis is crucial for combating the COVID-19 crisis. The knowledge acquired from decades of coronavirus research has provided a solid foundation for understanding COVID-19 and developing a response. As described in this review, multiple essential functions of the E protein have been discovered, which play a crucial role in the production of mature and competent virions. These functions include assembly, budding,critical roles in disease pathogenesis. However, despite significant advancements in its study, a great deal remains to be uncovered. Future understanding will undoubtedly elucidate new mechanisms of virology, host immune responses, and disease pathogenesis. Accordingly, identifying such mechanisms is essential to define therapeutic targets for this devastating pathogen and disease.
Acknowledgments
This work was supported by the Burroughs Wellcome Fund. Figures were created with Biorender.
Conflict of interest
The authors declare no conflict of interest.
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