COVID-19 Genomics: Study Shows Proteins Encoded By Open Reading Frame Segment ORF9c Of SARS-COV-2’s RNA Are Responsible For Immune Evasion

COVID-19 Genomics: Researchers from Sanford Burnham Prebys Medical Discovery Institute and the University of California-San Francisco have discovered that a protein encoded by the subgenomic sequence ORF9c of SARS-CoV-2 is responsible for the low antiviral response in infected humans, enabling the successful replication of the virus in the host. These proteins are also most probably responsible for the immune dysregulation in the human host.


 
The study findings are published on a preprint server is are pending peer review. https://www.biorxiv.org/content/10.1101/2020.08.18.256776v1
 
The detailed research utilized proteomics, interactome and transcriptional analysis passed through bioinformatics tools, to examine the effects of this small, 73-residue protein.
 
The research looked at the accessory viral protein ORF9c, which has been shown by earlier proteomic and interactome studies to affect the way the host cell operates through many cellular signaling pathways. These include immune signaling via interferons (IFNs) and interleukins (ILs).
 
It was found that this protein maintains the structure of organelles involved in viral replication as well as the virus itself, and the researchers show here that its sole expression alters cellular networks such that the outcome closely simulates full-fledged viral infection.
 
It has been found the ORF9c protein is a very unstable protein, found also in the earlier SARS coronavirus of 2003. It is also now known to play a role in causing the disease. Phylogeny and sequence alignment studies show that different coronaviruses have mutant variants of this protein, with the closest being in the bat coronavirus ORF14.
 
The detailed analysis of the protein predicts that it has a transmembrane sequence at the C-terminal region, unlike SARS-CoV. The reading frame can extend by three amino acids with a single nucleotide mutation. This makes SARS-CoV-2 ORF9c the only such protein in a human coronavirus to contain a transmembrane domain.
 
This unique ability to anchor itself to the host cell could increase its virulence and pathogenicity, by helping it to interact with the IFN pathway, changing antigen presentation, and immune evasion capabilities.
 
The study team also looked at the role of this protein in an epithelial lung cancer cell line. It interacts with multiple membrane-associated proteins scattered in many cellular compartments, as might be expected from the presence of the transmembrane domain, including those systems that involve protein synthesis and transport like the endoplasmic reticulum, Golgi apparatus, and mitochondria.
 
The study team discovered that different proteins were expressed in the transfected and untransfected samples, with the significant change being protein downregulation in 144 cases, and upregulation in 14 cases. The most significant change was in IFN signaling, antigen presentation, and innate immune pathways, including pattern recognition receptors. Proteins in the MAPK/ERK2 pathway were increased.
 
Significantly, the study team also found that small concentrations of ORF9c can induce changes in cellular pathways, inhibiting IFN, immune recognition, and ubiquitin components at the protein level, contributing to immune evasion.
 
The team went on to assess transcriptional changes due to ORF9c expression. They found a higher number of transcripts to be differentially regulated than proteins, but the pattern was similar in that most involved immune signaling. However, the specific pathways were different in many cases.
 
Importantly the biggest changes were on the complement and some inflammatory pathways, such as antigen presentation and immune signaling.
 
A significant observation is that some unique transcriptional changes were in the induction of IL-6 and p38 MAPK signaling pathways, not mirrored in proteomic analysis.
 
The team found that while both pathways showed the same pathways underwent changes in the same direction, the number of components affected in each pathway differed at protein and transcript level.
 
A detailed analysis of the interactions among the various components showed that downregulation was mainly observed concerning transcripts and proteins related to chemotaxis, complement, interferon, and antigen presentation.
Stress-related pathways and histone acetylation transcripts were induced by ORF9c, perhaps because transcriptional regression mediates some of the ORF9c-produced changes in gene expression.
 
Interestingly the addition of a proteasome inhibitor caused downregulation of some transcripts and proteins, but not all. The most significant change was with the ubiquitin pathway (UBP) components, and the unfolded protein response (UPR), which is vital for the regulation of cell cycle pathways. The researchers postulate that this may mean that these pathways take part in the breakdown of ORF9c. Again, it may mean that the ability to reduce important cell signaling pathways required for antiviral defenses depends on the presence of VCP.
 
The utilization of a different cell line may account for the differences between the findings of the current study and earlier research, which used HEK293 cells, as well as other criteria. The changes caused by exposure to this single protein are recapitulated in cells infected by the intact virus.
 
Also observed was that the greatest changes were in the loss of regulation of the IFN system, along with cytokines involved in the TNF and STAT signaling and innate immune factors. IFN signaling is involved in mounting an antiviral response, and in many of the disease features found in COVID-19, which can thus be traced back to the activity of this protein.


Interactome of ORF9c is based on LC-MS/MS of ORF9c-interacting proteins immunoprecipitated from A549 24 h after transfection. Left: Number of ORF9Cinteracting proteins according to their cell compartment (from Gene Ontology). Total values exceed 100% because some proteins are assigned / located in more than one compartment. Right: Protein subcellular localization map for the portion of the ORF9c interactome that is either a membrane protein or a membrane-related protein (as defined by Gene Ontology).
 
Corresponding author of the study, Dr Raffaella Pippa from the Cancer Center, Sanford Burnham Prebys Medical Discovery Institute told Thailand Medical News, “Our findings suggested that ORF9c enables cells to escape from immune surveillance through by reducing HLA abundance and antigen presentation, while also slowing cell replication, which could facilitate viral replication of infected cells.”
 
It should be noted that less than 2% of patients have a detrimental mutation in the transmembrane domain encoding sequence. This will need further study to understand how this affects clinical outcomes.
 
Also the link to histone acetylation may indicate a mechanism for transcriptional repression caused by this protein, including the upregulation of stress-induced genes following exposure to this viral protein. This could restrict immune signaling and promote immune evasion.
 
The ubiquitin pathway (UBP) association also harmonizes with that of cellular immune component downregulation, indicating that perhaps the former changes mediate the stability of other proteins, which in turn causes reduced cytokine signaling and decreased innate immune responses.
 
Unfolded protein response (UPR) components also destabilize the ORF9c protein, causing it to be recognized as a misfolded protein by the host cell. This protein binds to the UPR and the UBP, resulting in higher proteasome activity. They found that inhibitors of VCP, which mediates the engagement of the protein with the UPR, reduces the transcriptional repression effects of ORF9c on components of the immune system. Proteasomal inhibitors also have similar but less consistent effects.
 
The new study findings could also lead to successful inhibition of viral immune evasion mechanisms.
 
Past research has shown that such viral evasion mechanisms are a prime target for therapeutic intervention, to allow the host immune system to clear the virus more effectively. Most current vaccine candidates are directed at inhibiting viral entry via the spike protein, while remdesivir, an approved anti-SARS-CoV-2 drug, targets viral replication and assembly.
 
The study team says that further studies are needed to validate these findings in animal models.
 
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