Targeting viral protein ORF3a to fight Covid-19!

Monika R, PSG College of Technology, Coimbatore

The novel severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has caused a global pandemic of COVID-19 on an unprecedented scale. Coronaviruses may cause acute and chronic respiratory, enteric, and CNS infections. The presence of a large number of asymptomatic/mildly symptomatic patients, effective human-to-human transmission, and immunomodulatory characteristics of SARS-CoV-2 contribute to the current large-scale pandemic. Efforts are made for developing antivirals, immunomodulatory agents, vaccines, as well as rapid and accurate diagnostic tools. To tackle this viral disease, understanding the viral genome and evolution, viral pathogenesis and transmissibility, viral protein function and structure, is necessary for the successful development of effective therapeutics and vaccination. SARS-CoV-2 belongs to the subfamily Orthocoronavirinae under the Coronaviridae family and subgenus Sarbecovirus under the betacoronavirus genus and is closely associated with SARS-related-CoVs.

Genome organization of SARS-CoV-2

SARS-CoV-2 are single-stranded, positive-sense RNA viruses around 27-32 kb. SARS-CoV-2 genomic organisation encodes 16 non-structural proteins (NSP1−NSP16), 4 structural proteins including Spike (S), Membrane (M), Envelope (E) and Nucleocapsid (N) proteins and 6 accessory proteins [ORF3a (275aa), ORF6 (61aa), ORF7a (121aa), ORF7b (43aa), ORF8 (121aa) and, ORF10 (38aa)].

What does ORF3a protein do?

Among the accessory proteins, ORF3a is the largest in SARS-CoV-2 and regulates viral entry within the host and has immunogenic properties.

1. ORF3a can induce caspase-dependent apoptosis in cells by significantly elevating the percentage of cells with activated caspase-1. But ORF3a of SARS-CoV-2 has relatively weaker pro-apoptotic activity, contributing to asymptomatic infection, causing rapid transmission of the virus.
2. ORF3a also interferes with autophagosome–lysosome fusion leading SARS-CoV-2 to hijack host cells to escape degradation, which facilitates virus replication. 

3. ORF3a is additionally responsible for ion-channel formation and modulates the release of virus from the host cell.
4. Orf3a impacts the host immune system by activating IL-1β secretion that eventually activates NF-kB signaling and NLRP3 inflammasome and contributes to the generation of a cytokine storm and ultimately causes severe lung damage through cell pyroptosis and apoptosis.
5. ORF3a has unique pathogenic functions and its mutation rate directly correlates to the mortality rate.

Origin of ORF3a protein

Orf3a of SARS-CoV-2 shares significant similarities with membrane proteins from coronaviruses, suggesting a recent origin from a member of the M-protein family. This relationship is explained by two mechanisms: either there’s a protracted evolutionary history of the 2 genes with persistent gene loss in the other groups where Orf3a isn’t found, or Orf3a has emerged recently from CoV-M proteins and evolved rapidly and therefore the latter explanation is more favored.

Structure of ORF3a protein

ORF3a proteins are split into six domains. 

• Domain-I contains an N-terminus signal peptide for subcellular localization of ORF3a. 
• Domain-II contains TNF receptor-associated factor-3 (TRAF-3) binding motif which activates inflammasome. 
• Domain-III is for ion-channel activity and includes the Cysteine-rich domain (homodimerization of ORF3a protein responsible for tetramerization). 
• Domain-IV contains a caveolin binding motif that regulates viral uptake and trafficking of protein. 
• Domain-V contains a tyrosine-based sorting motif YXXϕ liable for transport (Golgi to the plasma membrane). 

Domain-VI has a SGD motif.

Cryo-EM to 2.1-Å resolution was used for structure determination of SARS-CoV-2 ORF3a protein in lipid nanodiscs. 

• 3a dimer structure includes N-terminus, C-terminus, and a brief cytoplasmic loop. 3a is approximately 70Å tall with transmembrane region (40-Å) and cytosolic domain (30Å). The transmembrane region consists of 3 helices per protomer with N-termini on the extracellular side and C-termini on the cytosolic side. In the extracellular region, the transmembrane helices form the circumference of an ellipse with TM1–3 of one protomer following TM1–3 of the second protomer in clockwise order. TM1–TM2 and TM2–TM3 are joined by intracellular and extracellular linkers, respectively. Each protomer chain forms a pair of opposing β-sheets packed against each other in an eight-stranded β-sandwich. The outer sheet is created by strands β1, β2, and β6 and half of β7 (N-terminal), and the inner sheet is made by strands β3, β4, β5, and β8 and half of β7 (C-terminal). The inner sheets from each protomer interact with other residues forming an eternal hydrophobic core.
• 3a tetramer structure shows the side-by-side arrangement of 2-dimers with separated TMs and proximity of CDs. A continuous interface is formed between TM3–CTD linkers and β1–β2 linkers from neighboring dimers. Tetramerization is mediated through hydrophobic, polar, and electrostatic interactions.

Ion conduction pathways are required for ion channel function, so 3a forms an outsized polar cavity within the TM region. The cavity is continuous with the cytosol and surrounding bilayer, through 3-pairs of openings: the upper (between TM2 and TM3), lower (underneath the TM1–TM2 linker, above CD), and intersubunit tunnels (between TM1 and TM3 from opposing protomers, above CD). While the cavity reduces the energetic barrier to ion movement across the inner half of the low dielectric membrane, a hydrophobic seal is created between TMs in the extracellular region above the polar cavity. So, opening a central pore would require conformational rearrangement to disrupt these hydrophobic interactions. Alternatively, lateral conduction pathways can be formed along conserved hydrophilic membrane-facing grooves between TM2 and TM3. The hydrophilic character of this region would be expected to lower the energetic barrier for the movement of ions across the outer half of the membrane. SARS-CoV-2 3a exhibits permeability to large cations including NMDG+ and YO-PRO-1 and sensitivity to ruthenium red and Gd3+.

ORF3a triggers calcium influx and programmed cell death in cells, which serve as a switch that activates calcium-dependent caspases and apoptosis. Thus, the expression of a calcium-permeable channel could affect lung homeostasis and COVID-19 pathogenesis.

ORF3a – A therapeutic target!

ORF3a is a key protein that can probably help to shed light on the pathogenicity of this deadly coronavirus. In-silico studies have concluded that the mutation rate of ORF3a directly correlates to the mortality rate. Thus, studying mutations in the ORF3a protein sequence becomes an important area in the control of virus infection. Coronavirus lineages that infect bats and humans are found with 3a like proteins, suggesting that targeting ORF3a could treat COVID-19 and it points the way to future experiments to illuminate the role of 3a in the viral life-cycle and disease pathology.

Also read: Flavor enhancers – An overview

References: 

• Kern, D.M., Sorum, B., Mali, S.S. et al. (2021). Cryo-EM structure of SARS-CoV-2 ORF3a in lipid nanodiscs. Nat Struct Mol Biol. doi.org/10.1038/s41594-021-00619-0
• Zhang, Y., Sun, H., Pei, R. et al. (2021). The SARS-CoV-2 protein ORF3a inhibits fusion of autophagosomes with lysosomes. Cell Discov 7, 31. doi: org/10.1038/s41421-021-00268-z
• Hassan, S. S., Attrish, D., Ghosh, S., Choudhury, P. P., & Roy, B. (2021). Pathogenic perspective of missense mutations of ORF3a protein of SARS-CoV-2. Virus research, 300, 198441. doi: org/10.1016/j.virusres.2021.198441
• Velazquez-Salinas, L., Zarate, S., Eberl, S., Gladue, D. P., Novella, I., & Borca, M. V. (2020). Positive Selection of ORF1ab, ORF3a, and ORF8 Genes Drives the Early Evolutionary Trends of SARS-CoV-2 During the 2020 COVID-19 Pandemic. Frontiers in Microbiology, 11. doi:10.3389/fmicb.2020.550674 
• Yang, S., Tian, M., & Johnson, A. N. (2020). SARS-CoV-2 protein ORF3a is pathogenic in Drosophila and causes phenotypes associated with COVID-19 post-viral syndrome. bioRxiv: the preprint server for biology, 2020.12.20.423533. doi: org/10.1101/2020.12.20.423533
• Majumdar, P., & Niyogi, S. (2020). ORF3a mutation associated with higher mortality rate in SARS-CoV-2 infection. Epidemiology and Infection, 148, E262. doi:10.1017/S0950268820002599
• Ren, Y., Shu, T., Wu, D. et al. (2020). The ORF3a protein of SARS-CoV-2 induces apoptosis in cells. Cell Mol Immunol 17, 881–883. doi: org/10.1038/s41423-020-0485-9
• Ouzounis C. A. (2020). A recent origin of Orf3a from M protein across the coronavirus lineage arising by sharp divergence. Computational and structural biotechnology journal, 18, 4093–4102. doi: org/10.1016/j.csbj.2020.11.047
• Lam, J. Y., Yuen, C. K., Ip, J. D., Wong, W. M., To, K. K., Yuen, K. Y., & Kok, K. H. (2020). Loss of orf3b in the circulating SARS-CoV-2 strains. Emerging microbes & infections, 9(1), 2685–2696. doi: org/10.1080/22221751.2020.1852892

About author:

Monika R is an enthusiastic Biotech student aspiring for an opportunity to develop skills and grow professionally in the research field. Extremely motivated and possess strong interpersonal skills and the ability to learn concepts quickly.

• The Corrosion Prediction from the Corrosion Product Performance
• Nitrogen Resilience in Waterlogged Soybean plants
• Cell Senescence in Type II Diabetes: Therapeutic Potential
• Transgene-Free Canker-Resistant Citrus sinensis with Cas12/RNP
• AI Literacy in Early Childhood Education: Challenges and Opportunities