Like a Swiss army knife, the SARS-CoV-2 accessory protein, ORF8, is multifunctional; may … [+]
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This article is an extension of our series on SARS-CoV-2 immunosuppression. The series has since been published as a book, Natural Immunity and Covid-19: What is and How it Can Save Your Life. It is also available to read on my website.
One of the keys to the rapid spread and global persistence of SARS-CoV-2 is its ability to evade and suppress our immune systems. Although other viruses also implement a variety of tactics and tricks, SARS-CoV-2 is particularly cunning. Researchers at the University of Pennsylvania Perelman School of Medicine, adding to a growing list, recently discovered another similar SARS-CoV-2 strategy: human histone protein mimicry. Published in Nature, Lee et al. show that one of the virus’s accessory proteins, ORF8, mimics the host histone H3 protein, interfering with cellular gene regulation and immune defense.
Most living cells carry chromosomes in their nucleus. These are thread-like structures made up of long, thin DNA fibers. They carry genetic information in the form of genes, allowing cells to copy themselves precisely during cell division. Importantly, the DNA strands are covered with packaging proteins known as histones (Figure 1). Think of these as genetic on/off switches: the tighter the histones are, the harder it is to read the DNA, and the looser the histones, the easier. This allows cells to regulate gene expression, including genes related to our immune response. Combined, the DNA strands and histone proteins are known as chromatin.
FIGURE 1. A schematic diagram representing the structure of a chromosome, including its components: DNA … [+]
SOURCE: National Human Genome Research Institute (Glossary: Histone)
Previous research had shown that SARS-CoV-2 can impair chromatin regulation, leading to reduced cellular antiviral responses. But the exact mechanism remains poorly understood.
Lee et al. suspicion of histone mimicry may play a role.
Master mimic: ORF8 mimics histone H3
To investigate their hunch, the scientists performed a bioinformatics comparison of all SARS-CoV-2 proteins with all human histone proteins. They found that a genetic sequence for the viral accessory protein ORF8 shared a striking similarity to a sequence found in the tail region of histone H3, one of the five major histones. The culprits? Six amino acid residues: alanine, arginine, lysine, serine, alanine, and proline (Figure 2). The first four of these were found to repeat at a second site within the histone H3 tail region.
FIGURE 2. ORF8 contains an ARKS motif at amino acid 50 that coincides with the tail of histone H3. Amino acids … [+]
FROM: “SARS-CoV-2 disrupts host epigenetic regulation through histone mimicry” LEE ET AL. 2022
The ARKS genetic motif is well known as an important site for gene regulation within histone H3. Remember that histones act as on/off switches for gene expression. Enzymes turn them on or off by adding molecular groups; Acetyl groups “turn on” DNA, methyl groups “turn off” DNA.
During viral infection, the immune system relies on such post-translational regulation to orchestrate an effective defensive response: modulating inflammation, recruiting the appropriate immune cells, etc. It interferes with histone-mediated gene expression and could interfere with antiviral responses. ORF8, by mimicking histone H3, could be doing just that.
Chromosomes are found in the nucleus of a cell. But it is rare that the proteins from SARS-CoV-2 and the proteins from the coronavirus as a whole can enter the nucleus. If ORF8 were mimicking histone H3, it would have to be able to get into the nucleus. To find out if ORF8 met this prerequisite, Lee and his colleagues exposed human cells to ORF8. Sure enough, they discovered that the viral protein could pass into the nucleus of the cells. Once inside, ORF8 interacted with chromatin and histone H3, modifying gene expression. The same ORF8 protein with the deleted ARKS motif also made it into the nucleus, but showed only a weakened ability to bind to chromatin.
The researchers noted that an enzyme called histone acetyltransferase KAT2A, commonly known simply as KAT2A, interacts with the ARKS motif of ORF8. Typically, this enzyme interacts with histone H3 by adding an acetyl group to the lysine amino acid in the ARKS motif, turning on certain genes. I was doing the same thing with ORF8’s ARKS motif. In fact, when ORF8 was added to cells, KAT2A levels dropped dramatically. Lee et al. speculate that ORF8 was degrading the enzyme after binding to it, preventing the enzyme from interacting with its intended target, histone H3.
KAT2A knockdown was associated with a reduction in active gene expression; the histone modifications that normally activate these genes were not occurring, as ORF8 successfully diverted trafficking. Instead, histones were packed more tightly, and DNA transcription, the process by which DNA is copied into RNA for protein production, was also decreased.
What about SARS-CoV-2?
OFR8 in isolation was having these effects, but would it have the same effect during live SARS-CoV-2 infection? To test this, the scientists infected human cells with wild-type SARS-CoV-2. As before, ORF8 succeeded in interfering with histone-associated gene expression. The modified SARS-CoV-2 that lacks the ORF8 gene, by contrast, failed to disrupt transcription. A version of the virus that contained the ORF8 gene, but modified in such a way that the ARKSAP motif was missing, also did not interfere with gene expression.
Compared with the wild-type strain, the strain that completely lacked ORF8 had a lower replication rate. Interestingly, the strain with the modified ORF8 matched the wild-type virus. This is in line with previous research indicating that ORF8 has a variety of different ways of helping SARS-CoV-2 to suppress and evade immunity.
In addition to affecting replication rates, the ORF8 modification also affects transcription within the host cell: the three strains induced different transcriptional responses, but only the wild-type strain succeeded in mimicking histone H3 and altering host cell chromatin. .
Transcendence
This work by Lee et al. contributes to a growing body of research on the immuno-evasive and immunosuppressive abilities of SARS-CoV-2. It should serve to remind us of two critical points.
First, while the spike protein is vital to infection—it is, after all, the main point of contact between the virus and the host cell—it is by no means the only relevant viral protein; SARS-CoV-2 has a large number of accessory proteins, many of which have tangible effects on the infection process. Lest we allow the virus to overtake us, we must keep ourselves well informed about the entire viral genome.
ORF8, in particular, has been shown to be a Swiss Army knife of SARS-CoV-2 immune obstruction. Aside from their role in histone mimicry, they also:
Stimulates lysosomal degradation of major histocompatibility complex type I (MHC-I) molecules – think of them as emergency buds that attach to the surface of cells, alerting nearby immune cells of infection. ORF8 hijacks the Beclin 1 autophagy initiation pathway, leading to MHC-I autophagy. As a result, infected cells remain invisible to the immune system, making them less likely to be killed and allowing for continued viral replication. It antagonizes the retinoic acid-inducible gene I (RIG-I) and the melanoma differentiation-associated protein-5 (MDA-5) signaling pathway by targeting HSP90B1, a chaperone protein that helps fold a variety of different receptors toll type (TLR). This inhibits the production of type 1 interferons (IFN-I), a set of signaling proteins that play a critical role in orchestrating immune responses. IFN-I downregulation helps enhance viral replication during the early stages of infection. It manages to escape degradation in the endoplasmic reticulum (ER) by forming disulfide complexes with the resident ER proteins. The endoplasmic reticulum is where newly synthesized proteins are folded into shape; during viral infection, it is a key component of virion assembly and maturation. By forming disulfide complexes, ORF8 reshapes the morphology of the endoplasmic reticulum, accelerating the transport of proteins from the endoplasmic reticulum to the Golgi apparatus. This can help speed up virion assembly and maturation. And finally, ORF8 mimics the host’s interleukin 17 (IL-17), a family of proinflammatory signaling proteins. ORF8 binds to the major interleukin 17 receptor, called the IL-17 receptor A (IL17RA), activating the IL-17 signaling pathway and triggering inflammation. ORF8-induced inflammation is a driving force for immune cell infiltration and lung injury, including fibrosis and coagulation dysregulation. SARS-CoV-2 variants lacking ORF8 are associated with milder infection and better disease outcome.
This brings us to the second important implication: to effectively disrupt a viral mechanism, you first need to understand it. The more we know about SARS-CoV-2, the better we can design drugs and therapeutic interventions. By expanding our knowledge of virus-host interactions, we begin to reveal areas of potential weakness, multiplying our targets.
Source: news.google.com