Covid variants

Troubling news out of Michigan

The Detroit News reported on 05 April 2021 that 246 of those vaccinated for Coivd-19 in Michigan later came down with a PCR positive infection. Some of these cases were severe enough to require hospitalization. Of these, three died. These cases were 14 or more days after the final dose in the series. The Detroit News did not state whether these new cases were the new variants that has Centers for Disease Control concerned.

Variants that concern CDC

The CDC has a list of Covid variants that it is concerned about or simply interested in. Most of the mutations in these variants of concern occur in the spike protein. The B.1.1.7 from the UK that was making the news of Meet the Press this morning has several amino acid substitutions as well as some single amino acid deletions in the spike protein according to this CDC site. This variant is more infectious but not less reactive with convalescent antibodies. P.1 from Japan/Brazil has shown “Reduced neutralization by convalescent and post-vaccination sera” Four of the five single amino acid substitutions of this variant occur in the amino acid 417-501 region of the spike protein. The same amino acid substitutions in the spike protein have been observed in the South African variant B.1.351. The CDC reports ~50% increased transmission and moderate reduction on neutralization by convalescent and post-vaccination sera. B.1.429, the US-California, variant has four amino acid substitutions in the spike protein. The CDC reports The CDC reports ~50% increased transmission and moderate reduction on neutralization by convalescent and post-vaccination sera. Mutations in the open reading frame (ORF) 1a and 1b were listed in strains of “interest.” The CDC didn’t say how these single amino acid substitutions could contribute to pathology of the RNA-dependent RNA polymerase protein product.

D614G, a perplexing paradigm shift

D416G is a biochemistry term using single letter codes for amino acids. The 614^th amino acid, aspartic acid (D), is replaced with glycine (G). This youtube video describes what was known about Covid-19 into mammalian cells at the start of the pandemic and before these new mutations emerged. Note that when the S1 domain of the spike protein binds to the ACE2 receptor the TMPRSS2 protease is activated. This cleaves off S1 allowing the S2 region to attach itself to the cell membrane.

All of the variants that that have the CDC concerned have a D614G mutation of the spike protein in which an aspartic acid is replaced with a glycine. This creates an arginine-glycine-aspartic acid RGD motif that cells use to bind to extracellular membrane integrins. Integrins, with α and β subunits, are trans membrane protein with intra- and extra-cellular domains. Integrins use the extracellular domains to attach to extracellular matrix proteins that contain the RGD motif. How does this new RGD site created by the D614G single amino acid substitution interact with other mutations in Covid-19 spike variants? Covid spike variant containing this RGD site and other mutations S477N, V483A, and N501Y were created in silico using an established X-ray crystal structure of the spike protein. [1] The authors ran computer simulations to hypothesize how these variants might bind the ACE2 receptor and Integrin receptors on cell surfaces. [1]

Integrin binding of the D614G in combo with other mutations…

The RGD sequence is located at 403–405 neighboring to the ACE-2 interaction site. [1] Jakhmola and coauthors discussed the use of the RGD motif by viruses to bind to cell surfaces. [1] They cited references that these integrin combinations may recognize the spike protein of Covid-19 with the RGD motif: α5β1, α8β1, αvβ1, αvβ3, αvβ8, αvβ5, αvβ6. Integrin heterodimers αbβ3, αvβ3, αvβ6 were predicted to be interact strongly with R403 and D405 of SARS-CoV-2 receptor binding domain. [1]

Integrin binding, so what? An hint from Herpes Simplex Virus (HSV)

αvβ8 and αvβ6 were shown to have a higher affinity for the HSV gH/gL glycoprotein than did the previously characterized αvβ3 receptor. [2] The authors found that integrin interaction enhanced but did not eliminate the need for the nectin receptor. [2] These authors also discussed the role of integrins, endocytosis, and lysosomal trafficking of HSV. [2]

Asking more questions with a brilliant review

Makowski and coauthors [3] published an excellent review on structure /function relationships of this new RGD motif in the Covid virus and what it means for receptor binding.

1. Covid binds to the integrin dimer, but is not internalized. Additionally it blocks the natural ligand from binding and hence acts as an antagonist.[3]
2. Covid binds to the integrin dimer, but is not internalized. It mimics the natural ligand causing down stream signaling events.
3. Covid is internalized and causes an infection.[3]
4. Covid is internalized and does not escape the endosome, but instead stays in a state of stasis. Our proposal.

Can a virus bind to both ACE2 and the the top annexin heterodimer, say αvβ5. What does αvβ5 do in pulmonary endothelial cells that also express ACE2? What happens when natural ligands bind to αvβ5? Su and coworkers have addressed this question as it pertains to acute lung injury that is characterized by increased vascular permeability. [4] Types of acute lung injury and their hormonal agents include (1) ventilation-induced lung injury partially mediated by transforming growth factor-β (TGF-β) and (2) ischemia-reperfusion mediated by vascular endothelial growth factor (VEGF). [4] These authors studied the response of αvβ5 to these hormones in cultured cells as well as “knock out” mice lacking the gene for the β5 subunit of αvβ5. [4] These authors grew pulmonary endothelial cells in cups with porous membranes on the bottom. (Fig 3A) These porous membranes were placed in wells with medium on the bottom. The top solution is a surrogate for blood whereas the bottom container is a surrogate for the tissue side. The top side was loaded with radioactive bovine serum albumin (BSA). Peptide hormones that bind to αvβ5 integrin were placed in the top container (Figure 3B). Most important to our story is the effect of the neutralizing antibody to αvβ5 studies they performed in cultured cells. When the cells round up, more BSA is able to pass into the lower camber (Figure 3B.

Integrin receptors are found on the bottom of the cells that help them attach to surfaces. Other integrin receptors are on the blood side and are there to tell the endothelial cell how to change in response to peptide hormones in the blood (Figure 3C. ) Stress fibers in cells are composed of actin and myosin filaments. Atin filaments may be viewed as ropes attached to cell surface contacts (Figure 3E). Myosin filaments are composed of myosin motors which can be thought of as people pulling on the rope (Figure 3D). Y-27632 is a small molecule inhibitor of rho associated protein kinase (ROCK) that in turn activates myosin motor activity.

Note that Y-27632, that ultimately inactivates myosin motors, prevents bovine serum albumin leakage across endothelial monolayers in response to all three ligands of αvβ5. [4] Also note: Su and coworkers also demonstrated that the antibody against αvβ5 prevented the increase in endothelial monolayer permeability in response to these three cytokines in human mouse endothelial cells . [4]

• Thrombin adds an interesting twist the the story of this new variety of Coivd-19. Thrombin is a protease that activates clotting cascades that are a problem in Covid
• VEGF is a growth factor that binds to receptors that are not integrins.
• TGFβ has its own receptor family separate from integrins. It also binds to blood clot degrading protease plasmin.

We still do not know how this D416G mutant variant will affect vascular permeability that is already a problem in Covid. From the appearance of things, it would make it worse.

References

1. Jakhmola, Shweta et al. “Mutational analysis of structural proteins of SARS-CoV-2.” Heliyon vol. 7,3 (2021): e06572. doi:10.1016/j.heliyon.2021.e06572 PMC free article
2. Gianni, T., Salvioli, S., Chesnokova, L. S., Hutt-Fletcher, L. M., & Campadelli-Fiume, G. (2013). αvβ6- and αvβ8-integrins serve as interchangeable receptors for HSV gH/gL to promote endocytosis and activation of membrane fusion. PLoS pathogens, 9(12), e1003806. https://doi.org/10.1371/journal.ppat.1003806 PMC free article
3. Makowski, L., Olson-Sidford, W., & W-Weisel, J. (2021). Biological and Clinical Consequences of Integrin Binding via a Rogue RGD Motif in the SARS CoV-2 Spike Protein. Viruses, 13(2), 146. https://doi.org/10.3390/v13020146 PMC free article
4. Su, G., Hodnett, M., Wu, N., Atakilit, A., Kosinski, C., Godzich, M., Huang, X. Z., Kim, J. K., Frank, J. A., Matthay, M. A., Sheppard, D., & Pittet, J. F. (2007). Integrin alphavbeta5 regulates lung vascular permeability and pulmonary endothelial barrier function. American journal of respiratory cell and molecular biology, 36(3), 377–386. https://doi.org/10.1165/rcmb.2006-0238OC PMC free article
5. Alam SB, Willows S, Kulka M, Sandhu JK. (2021) Severe acute respiratory syndrome coronavirus 2 may be an underappreciated pathogen of the central nervous system Eur J Neurol. 2020 Nov;27(11):2348-2360. Free PMC article.